Microorganisms for the production of 1,4-butanediol

ABSTRACT

The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO. The invention additionally provides methods of using such microbial organisms to produce BDO.

This application is a continuation of U.S. application Ser. No.13/449,187, filed Apr. 17, 2012, now U.S. Pat. No. 9,175,297, which is acontinuation of U.S. application Ser. No. 13/009,813, filed Jan. 19,2011, now U.S. Pat. No. 8,178,327, which is a continuation of U.S.application Ser. No. 12/947,790, filed Nov. 16, 2010, now U.S. Pat. No.8,129,156, which is a continuation of U.S. application Ser. No.12/556,550, filed Sep. 9, 2009, now U.S. Pat. No. 7,858,350, whichclaims the benefit of priority of U.S. provisional application Ser. No.61/191,710, filed Sep. 10, 2008, and U.S. provisional application Ser.No. 61/192,511, filed Sep. 17, 2008, each of which the entire contentsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates generally to in silico design of organisms and,more particularly to organisms having 1,4-butanediol biosynthesiscapability.

The compound 4-hydroxybutanoic acid (4-hydroxybutanoate,4-hydroxybutyrate, 4-HB) is a 4-carbon carboxylic acid that hasindustrial potential as a building block for various commodity andspecialty chemicals. In particular, 4-HB has the potential to serve as anew entry point into the 1,4-butanediol family of chemicals, whichincludes solvents, resins, polymer precursors, and specialty chemicals.1,4-Butanediol (BDO) is a polymer intermediate and industrial solventwith a global market of about 3 billion lb/year. BDO is currentlyproduced from petrochemical precursors, primarily acetylene, maleicanhydride, and propylene oxide.

For example, acetylene is reacted with 2 molecules of formaldehyde inthe Reppe synthesis reaction (Kroschwitz and Grant, Encyclopedia ofChem. Tech., John Wiley and Sons, Inc., New York (1999)), followed bycatalytic hydrogenation to form 1,4-butanediol. It has been estimatedthat 90% of the acetylene produced in the U.S. is consumed forbutanediol production. Alternatively, it can be formed by esterificationand catalytic hydrogenation of maleic anhydride, which is derived frombutane. Downstream, butanediol can be further transformed; for example,by oxidation to γ-butyrolactone, which can be further converted topyrrolidone and N-methyl-pyrrolidone, or hydrogenolysis totetrahydrofuran. These compounds have varied uses as polymerintermediates, solvents, and additives, and have a combined market ofnearly 2 billion lb/year.

It is desirable to develop a method for production of these chemicals byalternative means that not only substitute renewable for petroleum-basedfeedstocks, and also use less energy- and capital-intensive processes.The Department of Energy has proposed 1,4-diacids, and particularlysuccinic acid, as key biologically-produced intermediates for themanufacture of the butanediol family of products (DOE Report, “TopValue-Added Chemicals from Biomass”, 2004). However, succinic acid iscostly to isolate and purify and requires high temperatures andpressures for catalytic reduction to butanediol.

Thus, there exists a need for alternative means for effectivelyproducing commercial quantities of 1,4-butanediol and its chemicalprecursors. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF INVENTION

The invention provides non-naturally occurring microbial organismscontaining a 1,4-butanediol (BDO) pathway comprising at least oneexogenous nucleic acid encoding a BDO pathway enzyme expressed in asufficient amount to produce BDO. The invention additionally providesmethods of using such microbial organisms to produce BDO.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing biochemical pathways to4-hydroxybutyrate (4-HB) and to 1,4-butanediol production. The first 5steps are endogenous to E. coli, while the remainder can be expressedheterologously. Enzymes catalyzing the biosynthetic reactions are: (1)succinyl-CoA synthetase; (2) CoA-independent succinic semialdehydedehydrogenase; (3) α-ketoglutarate dehydrogenase; (4) glutamate:succinate semialdehyde transaminase; (5) glutamate decarboxylase; (6)CoA-dependent succinic semialdehyde dehydrogenase; (7)4-hydroxybutanoate dehydrogenase; (8) α-ketoglutarate decarboxylase; (9)4-hydroxybutyryl CoA:acetyl-CoA transferase; (10) butyrate kinase; (11)phosphotransbutyrylase; (12) aldehyde dehydrogenase; (13) alcoholdehydrogenase.

FIG. 2 is a schematic diagram showing homoserine biosynthesis in E.coli.

FIGS. 3A-3C show the production of 4-HB in glucose minimal medium usingE. coli strains harboring plasmids expressing various combinations of4-HB pathway genes. FIG. 3A, 4-HB concentration in culture broth; FIG.3B, succinate concentration in culture broth; FIG. 3C, culture OD,measured at 600 nm. Clusters of bars represent the 24 hour, 48 hour, and72 hour (if measured) timepoints. The codes along the x-axis indicatethe strain/plasmid combination used. The first index refers to the hoststrain: 1, MG1655 lacI^(Q); 2, MG1655 ΔgabD lacI^(Q); 3, MG1655 ΔgabDΔaldA lacI^(Q). The second index refers to the plasmid combination used:1, pZE13-0004-0035 and pZA33-0036; 2, pZE13-0004-0035 and pZA33-0010n;3, pZE13-0004-0008 and pZA33-0036; 4, pZE13-0004-0008 and pZA33-0010n;5, Control vectors pZE13 and pZA33.

FIG. 4 shows the production of 4-HB from glucose in E. coli strainsexpressing α-ketoglutarate decarboxylase from Mycobacteriumtuberculosis. Strains 1-3 contain pZE13-0032 and pZA33-0036. Strain 4expresses only the empty vectors pZE13 and pZA33. Host strains are asfollows: 1 and 4, MG1655 lacI^(Q); 2, MG1655 ΔgabD lacI^(Q); 3, MG1655ΔgabD ΔaldA lacI^(Q). The bars refer to concentration at 24 and 48hours.

FIG. 5 shows the production of BDO from 10 mM 4-HB in recombinant E.coli strains. Numbered positions correspond to experiments with MG1655lacI^(Q) containing pZA33-0024, expressing cat2 from P. gingivalis, andthe following genes expressed on pZE13: 1, none (control); 2, 0002; 3,0003; 4, 0003n; 5, 0011; 6, 0013; 7, 0023; 8, 0025; 9, 0008n; 10, 0035.Gene numbers are defined in Table 6. For each position, the bars referto aerobic, microaerobic, and anaerobic conditions, respectively.Microaerobic conditions were created by sealing the culture tubes butnot evacuating them.

FIGS. 6A-6H show the mass spectrum of 4-HB and BDO produced by MG1655lacI^(Q) pZE13-0004-0035-0002 pZA33-0034-0036 grown in M9 minimal mediumsupplemented with 4 g/L unlabeled glucose (FIGS. 6A, 6C, 6E and 6G)uniformly labeled ¹³C-glucose (FIGS. 6B, 6D, 6F, and 6H). FIGS. 6A and6B, mass 116 characteristic fragment of derivatized BDO, containing 2carbon atoms; FIGS. 6C and 6D, mass 177 characteristic fragment ofderivatized BDO, containing 1 carbon atom; FIGS. 6E and 6F, mass 117characteristic fragment of derivatized 4-HB, containing 2 carbon atoms;FIGS. 6G and 6H, mass 233 characteristic fragment of derivatized 4-HB,containing 4 carbon atoms.

FIG. 7 is a schematic process flow diagram of bioprocesses for theproduction of γ-butyrolactone. Upper panel illustrates fed-batchfermentation with batch separation and lower panel illustrates fed-batchfermentation with continuous separation.

FIGS. 8A and 8B show exemplary 1,4-butanediol (BDO) pathways. FIG. 8Ashows BDO pathways from succinyl-CoA. FIG. 8B shows BDO pathways fromalpha-ketoglutarate.

FIGS. 9A-9C show exemplary BDO pathways. FIGS. 9A and 9B show pathwaysfrom 4-aminobutyrate. FIG. 9C shows a pathway from acetoacetyl-CoA to4-aminobutyrate.

FIG. 10 shows exemplary BDO pathways from alpha-ketoglutarate.

FIG. 11 shows exemplary BDO pathways from glutamate.

FIG. 12 shows exemplary BDO pathways from acetoacetyl-CoA.

FIG. 13 shows exemplary BDO pathways from homoserine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities for4-hydroxybutanoic acid (4-HB), γ-butyrolactone and 1,4-butanediol (BDO).The invention, in particular, relates to the design of microbialorganisms capable of producing BDO by introducing one or more nucleicacids encoding a BDO pathway enzyme.

In one embodiment, the invention utilizes in silico stoichiometricmodels of Escherichia coli metabolism that identify metabolic designsfor biosynthetic production of 4-hydroxybutanoic acid (4-HB) and1,4-butanediol (BDO). The results described herein indicate thatmetabolic pathways can be designed and recombinantly engineered toachieve the biosynthesis of 4-HB and downstream products such as1,4-butanediol in Escherichia coli and other cells or organisms.Biosynthetic production of 4-HB, for example, for the in silico designscan be confirmed by construction of strains having the designedmetabolic genotype. These metabolically engineered cells or organismsalso can be subjected to adaptive evolution to further augment 4-HBbiosynthesis, including under conditions approaching theoretical maximumgrowth.

In certain embodiments, the 4-HB biosynthesis characteristics of thedesigned strains make them genetically stable and particularly useful incontinuous bioprocesses. Separate strain design strategies wereidentified with incorporation of different non-native or heterologousreaction capabilities into E. coli or other host organisms leading to4-HB and 1,4-butanediol producing metabolic pathways from eitherCoA-independent succinic semialdehyde dehydrogenase, succinyl-CoAsynthetase and CoA-dependent succinic semialdehyde dehydrogenase, orglutamate: succinic semialdehyde transaminase. In silico metabolicdesigns were identified that resulted in the biosynthesis of 4-HB inboth E. coli and yeast species from each of these metabolic pathways.The 1,4-butanediol intermediate γ-butyrolactone can be generated inculture by spontaneous cyclization under conditions at pH<7.5,particularly under acidic conditions, such as below pH 5.5, for example,pH<7, pH<6.5, pH<6, and particularly at pH<5.5 or lower.

Strains identified via the computational component of the platform canbe put into actual production by genetically engineering any of thepredicted metabolic alterations which lead to the biosyntheticproduction of 4-HB, 1,4-butanediol or other intermediate and/ordownstream products. In yet a further embodiment, strains exhibitingbiosynthetic production of these compounds can be further subjected toadaptive evolution to further augment product biosynthesis. The levelsof product biosynthesis yield following adaptive evolution also can bepredicted by the computational component of the system.

In other specific embodiments, microbial organisms were constructed toexpress a 4-HB biosynthetic pathway encoding the enzymatic steps fromsuccinate to 4-HB and to 4-HB-CoA. Co-expression of succinate coenzyme Atransferase, CoA-dependent succinic semialdehyde dehydrogenase,NAD-dependent 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyratecoenzyme A transferase in a host microbial organism resulted insignificant production of 4-HB compared to host microbial organismslacking a 4-HB biosynthetic pathway. In a further specific embodiment,4-HB-producing microbial organisms were generated that utilizedα-ketoglutarate as a substrate by introducing nucleic acids encodingα-ketoglutarate decarboxylase and NAD-dependent 4-hydroxybutyratedehydrogenase.

In another specific embodiment, microbial organisms containing a1,4-butanediol (BDO) biosynthetic pathway were constructed thatbiosynthesized BDO when cultured in the presence of 4-HB. The BDObiosynthetic pathway consisted of a nucleic acid encoding either amultifunctional aldehyde/alcohol dehydrogenase or nucleic acids encodingan aldehyde dehydrogenase and an alcohol dehydrogenase. To supportgrowth on 4-HB substrates, these BDO-producing microbial organisms alsoexpressed 4-hydroxybutyrate CoA transferase or 4-butyrate kinase inconjunction with phosphotranshydroxybutyrlase. In yet a further specificembodiment, microbial organisms were generated that synthesized BDOthrough exogenous expression of nucleic acids encoding a functional 4-HBbiosynthetic pathway and a functional BDO biosynthetic pathway. The 4-HBbiosynthetic pathway consisted of succinate coenzyme A transferase,CoA-dependent succinic semialdehyde dehydrogenase, NAD-dependent4-hydroxybutyrate dehydrogenase and 4-hydroxybutyrate coenzyme Atransferase. The BDO pathway consisted of a multifunctionalaldehyde/alcohol dehydrogenase. Further described herein are additionalpathways for production of BDO (see FIGS. 8-13).

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes or proteins within a biosyntheticpathway for a BDO family of compounds.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “4-hydroxybutanoic acid” is intended to mean a4-hydroxy derivative of butyric acid having the chemical formula C₄H₈O₃and a molecular mass of 104.11 g/mol (126.09 g/mol for its sodium salt).The chemical compound 4-hydroxybutanoic acid also is known in the art as4-HB, 4-hydroxybutyrate, gamma-hydroxybutyric acid or GHB. The term asit is used herein is intended to include any of the compound's varioussalt forms and include, for example, 4-hydroxybutanoate and4-hydroxybutyrate. Specific examples of salt forms for 4-HB includesodium 4-HB and potassium 4-HB. Therefore, the terms 4-hydroxybutanoicacid, 4-HB, 4-hydroxybutyrate, 4-hydroxybutanoate, gamma-hydroxybutyricacid and GHB as well as other art recognized names are used synonymouslyherein.

As used herein, the term “monomeric” when used in reference to 4-HB isintended to mean 4-HB in a non-polymeric or underivatized form. Specificexamples of polymeric 4-HB include poly-4-hydroxybutanoic acid andcopolymers of, for example, 4-HB and 3-HB. A specific example of aderivatized form of 4-HB is 4-HB-CoA. Other polymeric 4-HB forms andother derivatized forms of 4-HB also are known in the art.

As used herein, the term “γ-butyrolactone” is intended to mean a lactonehaving the chemical formula C₄H₆O₂ and a molecular mass of 86.089 g/mol.The chemical compound γ-butyrolactone also is know in the art as GBL,butyrolactone, 1,4-lactone, 4-butyrolactone, 4-hydroxybutyric acidlactone, and gamma-hydroxybutyric acid lactone. The term as it is usedherein is intended to include any of the compound's various salt forms.

As used herein, the term “1,4-butanediol” is intended to mean an alcoholderivative of the alkane butane, carrying two hydroxyl groups which hasthe chemical formula C₄H₁₀O₂ and a molecular mass of 90.12 g/mol. Thechemical compound 1,4-butanediol also is known in the art as BDO and isa chemical intermediate or precursor for a family of compounds referredto herein as BDO family of compounds.

As used herein, the term “tetrahydrofuran” is intended to mean aheterocyclic organic compound corresponding to the fully hydrogenatedanalog of the aromatic compound furan which has the chemical formulaC₄H₈O and a molecular mass of 72.11 g/mol. The chemical compoundtetrahydrofuran also is known in the art as THF, tetrahydrofuran,1,4-epoxybutane, butylene oxide, cyclotetramethylene oxide,oxacyclopentane, diethylene oxide, oxolane, furanidine, hydrofuran,tetra-methylene oxide. The term as it is used herein is intended toinclude any of the compound's various salt forms.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

The non-naturally occurring microbial organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein are described withreference to a suitable host or source organism such as E. coli, yeast,or other organisms disclosed herein and their corresponding metabolicreactions or a suitable source organism for desired genetic materialsuch as genes encoding enzymes for their corresponding metabolicreactions. However, given the complete genome sequencing of a widevariety of organisms and the high level of skill in the area ofgenomics, those skilled in the art will readily be able to apply theteachings and guidance provided herein to essentially all otherorganisms. For example, the E. coli metabolic alterations exemplifiedherein can readily be applied to other species by incorporating the sameor analogous encoding nucleic acid from species other than thereferenced species. Such genetic alterations include, for example,genetic alterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the growth-coupledproduction of a biochemical product, those skilled in the art willunderstand that the orthologous gene harboring the metabolic activity tobe introduced or disrupted is to be chosen for construction of thenon-naturally occurring microorganism. An example of orthologsexhibiting separable activities is where distinct activities have beenseparated into distinct gene products between two or more species orwithin a single species. A specific example is the separation ofelastase proteolysis and plasminogen proteolysis, two types of serineprotease activity, into distinct molecules as plasminogen activator andelastase. A second example is the separation of mycoplasma 5′-3′exonuclease and Drosophila DNA polymerase III activity. The DNApolymerase from the first species can be considered an ortholog toeither or both of the exonuclease or the polymerase from the secondspecies and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having 4-HB, GBL and/or BDObiosynthetic capability, those skilled in the art will understand withapplying the teaching and guidance provided herein to a particularspecies that the identification of metabolic modifications can includeidentification and inclusion or inactivation of orthologs. To the extentthat paralogs and/or nonorthologous gene displacements are present inthe referenced microorganism that encode an enzyme catalyzing a similaror substantially similar metabolic reaction, those skilled in the artalso can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

The invention provides a non-naturally occurring microbial biocatalystincluding a microbial organism having a 4-hydroxybutanoic acid (4-HB)biosynthetic pathway that includes at least one exogenous nucleic acidencoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinicsemialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependentsuccinic semialdehyde dehydrogenase, glutamate: succinic semialdehydetransaminase, alpha-ketoglutarate decarboxylase, or glutamatedecarboxylase, wherein the exogenous nucleic acid is expressed insufficient amounts to produce monomeric 4-hydroxybutanoic acid (4-HB).4-hydroxybutanoate dehydrogenase is also referred to as4-hydroxybutyrate dehydrogenase or 4-HB dehydrogenase. Succinyl-CoAsynthetase is also referred to as succinyl-CoA synthase or succinyl-CoAligase.

Also provided is a non-naturally occurring microbial biocatalystincluding a microbial organism having a 4-hydroxybutanoic acid (4-HB)biosynthetic pathway having at least one exogenous nucleic acid encoding4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependentsuccinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase,wherein the exogenous nucleic acid is expressed in sufficient amounts toproduce monomeric 4-hydroxybutanoic acid (4-HB).

The non-naturally occurring microbial biocatalysts of the inventioninclude microbial organisms that employ combinations of metabolicreactions for biosynthetically producing the compounds of the invention.The biosynthesized compounds can be produced intracellularly and/orsecreted into the culture medium. Exemplary compounds produced by thenon-naturally occurring microorganisms include, for example,4-hydroxybutanoic acid, 1,4-butanediol and γ-butyrolactone.

In one embodiment, a non-naturally occurring microbial organism isengineered to produce 4-HB. This compound is one useful entry point intothe 1,4-butanediol family of compounds. The biochemical reactions forformation of 4-HB from succinate, from succinate through succinyl-CoA orfrom α-ketoglutarate are shown in steps 1-8 of FIG. 1.

It is understood that any combination of appropriate enzymes of a BDOpathway can be used so long as conversion from a starting component tothe BDO product is achieved. Thus, it is understood that any of themetabolic pathways disclosed herein can be utilized and that it is wellunderstood to those skilled in the art how to select appropriate enzymesto achieve a desired pathway, as disclosed herein.

In another embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having a1,4-butanediol (BDO) pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, the BDO pathway comprising 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoAtransaminase, or 4-hydroxybutyryl-CoA dehydrogenase (see Example VIITable 17). The BDO pathway further can comprise 4-hydroxybutyryl-CoAreductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or1,4-butanediol dehydrogenase.

It is understood by those skilled in the art that various combinationsof the pathways can be utilized, as disclosed herein. For example, in anon-naturally occurring microbial organism, the nucleic acids can encode4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, or4-aminobutyrate-CoA ligase; 4-aminobutyryl-CoA oxidoreductase(deaminating) or 4-aminobutyryl-CoA transaminase; and4-hydroxybutyryl-CoA dehydrogenase. Other exemplary combinations arespecifically describe below and further can be found in FIGS. 8-13. Forexample, the BDO pathway can further comprise 4-hydroxybutyryl-CoAreductase (alcohol forming), 4-hydroxybutyryl-CoA reductase, or1,4-butanediol dehydrogenase.

The invention additionally provides a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoAhydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase(alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-oldehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) or4-aminobutan-1-ol transaminase (see Example VII and Table 18), and canfurther comprise 1,4-butanediol dehydrogenase. For example, theexogenous nucleic acids can encode 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, or 4-aminobutyrate-CoA ligase;4-aminobutyryl-CoA reductase (alcohol forming); and 4-aminobutan-1-oloxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase. Inaddition, the nucleic acids can encode. 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, or 4-aminobutyrate-CoA ligase;4-aminobutyryl-CoA reductase; 4-aminobutan-1-ol dehydrogenase; and4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-oltransaminase.

The invention further provides a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising 4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase(phosphorylating), 4-aminobutan-1-ol dehydrogenase,4-aminobutan-1-oloxidoreductase (deaminating), 4-aminobutan-1-oltransaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase(deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase,4-hydroxybutyryl-phosphate dehydrogenase, or 4-hydroxybutyraldehydedehydrogenase (phosphorylating) (see Example VII and Table 19). Forexample, the exogenous nucleic acids can encode 4-aminobutyrate kinase;4-aminobutyraldehyde dehydrogenase (phosphorylating); 4-aminobutan-1-oldehydrogenase; and 4-aminobutan-1-ol oxidoreductase (deaminating) or4-aminobutan-1-ol transaminase. Alternatively, the exogenous nucleicacids can encode 4-aminobutyrate kinase;[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) or[(4-aminobutanolyl)oxy]phosphonic acid transaminase;4-hydroxybutyryl-phosphate dehydrogenase; and 4-hydroxybutyraldehydedehydrogenase (phosphorylating).

Also provided is a non-naturally occurring microbial organism,comprising a microbial organism having a BDO pathway comprising at leastone exogenous nucleic acid encoding a BDO pathway enzyme expressed in asufficient amount to produce BDO, the BDO pathway comprisingalpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehydedehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase,alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase,5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoAreductase (alcohol forming), 5-hydroxy-2-oxopentanoic aciddecarboxylase, or 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation) (see Example VIII and Table 20). The BDO pathway canfurther comprise 4-hydroxybutyryl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA reductase, or 1,4-butanediol dehydrogenase. Forexample, the exogenous nucleic acids can encode alpha-ketoglutarate5-kinase; 2,5-dioxopentanoic semialdehyde dehydrogenase(phosphorylating); 2,5-dioxopentanoic acid reductase; and5-hydroxy-2-oxopentanoic acid decarboxylase. Alternatively, theexogenous nucleic acids can encode alpha-ketoglutarate 5-kinase;2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);2,5-dioxopentanoic acid reductase; and 5-hydroxy-2-oxopentanoic aciddehydrogenase (decarboxylation). Alternatively, the exogenous nucleicacids can encode alpha-ketoglutarate CoA transferase,alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic aciddehydrogenase; and 5-hydroxy-2-oxopentanoic acid decarboxylase. Inanother embodiment, the exogenous nucleic acids can encodealpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA reductase,5-hydroxy-2-oxopentanoic acid dehydrogenase, and5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).Alternatively, the exogenous nucleic acids can encodealpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA reductase(alcohol forming); and 5-hydroxy-2-oxopentanoic acid decarboxylase. Inyet another embodiment, the exogenous nucleic acids can encodealpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase,or alpha-ketoglutaryl-CoA ligase; alpha-ketoglutaryl-CoA reductase(alcohol forming); and 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation).

The invention additionally provides a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising glutamate CoA transferase, glutamyl-CoA hydrolase,glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehydedehydrogenase (phosphorylating), glutamyl-CoA reductase,glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcoholforming), 2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating),2-amino-5-hydroxypentanoic acid transaminase, 5-hydroxy-2-oxopentanoicacid decarboxylase, 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation) (see Example IX and Table 21). For example, theexogenous nucleic acids can encode glutamate CoA transferase,glutamyl-CoA hydrolase, or glutamyl-CoA ligase; glutamyl-CoA reductase;glutamate-5-semialdehyde reductase; 2-amino-5-hydroxypentanoic acidoxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acidtransaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation).Alternatively, the exogenous nucleic acids can encode glutamate5-kinase; glutamate-5-semialdehyde dehydrogenase (phosphorylating);glutamate-5-semialdehyde reductase; 2-amino-5-hydroxypentanoic acidoxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acidtransaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). In stillanother embodiment, the exogenous nucleic acids can encode glutamate CoAtransferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;glutamyl-CoA reductase (alcohol forming); 2-amino-5-hydroxypentanoicacid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acidtransaminase; and 5-hydroxy-2-oxopentanoic acid decarboxylase or5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation). In yetanother embodiment, the exogenous nucleic acids can encode glutamate5-kinase; glutamate-5-semialdehyde dehydrogenase (phosphorylating);2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or2-amino-5-hydroxypentanoic acid transaminase; and5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation).

Additionally provided is a non-naturally occurring microbial organism,comprising a microbial organism having a BDO pathway comprising at leastone exogenous nucleic acid encoding a BDO pathway enzyme expressed in asufficient amount to produce BDO, the BDO pathway comprising3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase,vinylacetyl-CoA Δ-isomerase, or 4-hydroxybutyryl-CoA dehydratase (seeExample X and Table 22). For example, the exogenous nucleic acids canencode 3-hydroxybutyryl-CoA dehydrogenase; 3-hydroxybutyryl-CoAdehydratase; vinylacetyl-CoA Δ-isomerase; and 4-hydroxybutyryl-CoAdehydratase.

In another embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising homoserine deaminase, homoserine CoA transferase,homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoAdeaminase, 4-hydroxybut-2-enoyl-CoA transferase,4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase,4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or4-hydroxybut-2-enoyl-CoA reductase (see Example XI and Table 23). Forexample, the exogenous nucleic acids can encode homoserine deaminase;4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoAhydrolase, 4-hydroxybut-2-enoyl-CoA ligase; 4-hydroxybut-2-enoyl-CoAreductase. Alternatively, the exogenous nucleic acids can encodehomoserine CoA transferase, homoserine-CoA hydrolase, or homoserine-CoAligase; homoserine-CoA deaminase; and 4-hydroxybut-2-enoyl-CoAreductase. In a further embodiment, the exogenous nucleic acids canencode homoserine deaminase; 4-hydroxybut-2-enoate reductase; and4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, or4-hydroxybutyryl-CoA ligase. Alternatively, the exogenous nucleic acidscan encode homoserine CoA transferase, homoserine-CoA hydrolase, orhomoserine-CoA ligase; homoserine-CoA deaminase; and4-hydroxybut-2-enoyl-CoA reductase.

Further provided by the invention is a non-naturally occurring microbialorganism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BOD, the BDO pathwaycomprising succinyl-CoA reductase (alcohol forming),4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,4-hydroxybutanal dehydrogenase (phosphorylating)(also referred to hereinas acylphosphate reductase) (see Table 15). Such a BDO pathway canfurther comprise succinyl-CoA reductase, 4-hydroxybutyratedehydrogenase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyratekinase, phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoAreductase, 4-hydroxybutyryl-CoA reductase (alcohol forming), or1,4-butanediol dehydrogenase.

In another embodiment, the invention provides a non-naturally occurringmicrobial organism, comprising a microbial organism having a BDO pathwaycomprising at least one exogenous nucleic acid encoding a BDO pathwayenzyme expressed in a sufficient amount to produce BDO, the BDO pathwaycomprising glutamate dehydrogenase, 4-aminobutyrate oxidoreductase(deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,4-hydroxybutanal dehydrogenase (phosphorylating)(acylphosphatereductase)(see Table 16). Such a BDO pathway can further comprisealpha-ketoglutarate decarboxylase, 4-hydroxybutyrate dehydrogenase,4-hydroxybutyryl-CoA transferase, 4-hydroxybutyrate kinase,phosphotrans-4-hydroxybutyrylase, 4-hydroxybutyryl-CoA reductase,4-hydroxybutyryl-CoA reductase (alcohol forming), or 1,4-butanedioldehydrogenase.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 4-HB or BDO pathway, wherein thenon-naturally occurring microbial organism comprises at least oneexogenous nucleic acid encoding an enzyme or protein that converts asubstrate to a product in a 4-HB or BDO pathway, for example,succinyl-CoA to succinic semialdehyde, succinic semialdehyde to4-hydroxybutyrate, 4-hydroxybutyrate to 4-hydroxybutyryl-CoA,4-hydroxybutyryl-CoA to 4-hydroxybutyraldehyde, 4-hydroxybutyraldehydeto 1,4-butanediol, as exemplified in FIG. 1. In another embodiment, asubstrate to product in a 4-HB or BDO pathway can be, for example,succinyl-CoA to succinic semialdehyde, succinic semialdehyde to4-hydroxybutyrate, 4-hydroxybutyrate to 4-hydroxybutyryl-phosphate,4-hydroxybutyryl-phosphate to 4-hydroxybutyryl-CoA, 4-hydroxybutyryl-CoAto 4-hydroxybutanal, 4-hydroxybutanal to 1,4-butanediol, as exemplifiedin one embodiment of FIG. 8A. Thus, the invention provides anon-naturally occurring microbial organism containing at least oneexogenous nucleic acid encoding an enzyme or protein, where the enzymeor protein converts the substrates and products of a 4-HB or BDOpathway, such as those shown in FIGS. 8-13, and one skilled in the artcan readily determine such substrates and products based on the 4-HB orBDO pathways disclosed herein.

The pathways described above are merely exemplary. One skilled in theart can readily select appropriate pathways from those disclosed hereinto obtain a suitable 4-HB or BDO pathway or other metabolic pathway, asdesired.

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction and that reference toany of these metabolic constituents also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes as well as the reactantsand products of the reaction.

The production of 4-HB via biosynthetic modes using the microbialorganisms of the invention is particularly useful because it can producemonomeric 4-HB. The non-naturally occurring microbial organisms of theinvention and their biosynthesis of 4-HB and BDO family compounds alsois particularly useful because the 4-HB product can be (1) secreted; (2)can be devoid of any derivatizations such as Coenzyme A; (3) avoidsthermodynamic changes during biosynthesis; (4) allows directbiosynthesis of BDO, and (5) allows for the spontaneous chemicalconversion of 4-HB to γ-butyrolactone (GBL) in acidic pH medium. Thislatter characteristic also is particularly useful for efficient chemicalsynthesis or biosynthesis of BDO family compounds such as 1,4-butanedioland/or tetrahydrofuran (THF), for example.

Microbial organisms generally lack the capacity to synthesize 4-HB. Anyof the compounds disclosed herein to be within the 1,4-butanediol familyof compounds or known by those in the art to be within the1,4-butanediol family of compounds are considered to be within the1,4-butanediol family of compounds. Moreover, organisms having all ofthe requisite metabolic enzymatic capabilities are not known to produce4-HB from the enzymes described and biochemical pathways exemplifiedherein. Rather, with the possible exception of a few anaerobicmicroorganisms described further below, the microorganisms having theenzymatic capability use 4-HB as a substrate to produce, for example,succinate. In contrast, the non-naturally occurring microbial organismsof the invention can generate 4-HB or BDO as a product. As describedabove, the biosynthesis of 4-HB in its monomeric form is not onlyparticularly useful in chemical synthesis of BDO family of compounds, italso allows for the further biosynthesis of BDO family compounds andavoids altogether chemical synthesis procedures.

The non-naturally occurring microbial organisms of the invention thatcan produce 4-HB or BDO are produced by ensuring that a host microbialorganism includes functional capabilities for the complete biochemicalsynthesis of at least one 4-HB or BDO biosynthetic pathway of theinvention. Ensuring at least one requisite 4-HB or BDO biosyntheticpathway confers 4-HB biosynthesis capability onto the host microbialorganism.

Five 4-HB biosynthetic pathways are exemplified herein and shown forpurposes of illustration in FIG. 1. Additional 4-HB and BDO pathways aredescribed in FIGS. 8-13. One 4-HB biosynthetic pathway includes thebiosynthesis of 4-HB from succinate (the succinate pathway). The enzymesparticipating in this 4-HB pathway include CoA-independent succinicsemialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase. In thispathway, CoA-independent succinic semialdehyde dehydrogenase catalyzesthe reverse reaction to the arrow shown in FIG. 1. Another 4-HBbiosynthetic pathway includes the biosynthesis from succinate throughsuccinyl-CoA (the succinyl-CoA pathway). The enzymes participating inthis 4-HB pathway include succinyl-CoA synthetase, CoA-dependentsuccinic semialdehyde dehydrogenase and 4-hydroxybutanoatedehydrogenase. Three other 4-HB biosynthetic pathways include thebiosynthesis of 4-HB from α-ketoglutarate (the α-ketoglutaratepathways). Hence, a third 4-HB biosynthetic pathway is the biosynthesisof succinic semialdehyde through glutamate: succinic semialdehydetransaminase, glutamate decarboxylase and 4-hydroxybutanoatedehydrogenase. A fourth 4-HB biosynthetic pathway also includes thebiosynthesis of 4-HB from α-ketoglutarate, but utilizes α-ketoglutaratedecarboxylase to catalyze succinic semialdehyde synthesis.4-hydroxybutanoate dehydrogenase catalyzes the conversion of succinicsemialdehyde to 4-HB. A fifth 4-HB biosynthetic pathway includes thebiosynthesis from α-ketoglutarate through succinyl-CoA and utilizesα-ketoglutarate dehydrogenase to produce succinyl-CoA, which funnelsinto the succinyl-CoA pathway described above. Each of these 4-HBbiosynthetic pathways, their substrates, reactants and products aredescribed further below in the Examples. As described herein, 4-HB canfurther be biosynthetically converted to BDO by inclusion of appropriateenzymes to produce BDO (see Example). Thus, it is understood that a 4-HBpathway can be used with enzymes for converting 4-HB to BDO to generatea BDO pathway.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes participating in one or more 4-HB or BDO biosyntheticpathways. Depending on the host microbial organism chosen forbiosynthesis, nucleic acids for some or all of a particular 4-HB or BDObiosynthetic pathway can be expressed. For example, if a chosen host isdeficient in one or more enzymes in a desired biosynthetic pathway, forexample, the succinate to 4-HB pathway, then expressible nucleic acidsfor the deficient enzyme(s), for example, both CoA-independent succinicsemialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase in thisexample, are introduced into the host for subsequent exogenousexpression. Alternatively, if the chosen host exhibits endogenousexpression of some pathway enzymes, but is deficient in others, then anencoding nucleic acid is needed for the deficient enzyme(s) to achieve4-HB or BDO biosynthesis. For example, if the chosen host exhibitsendogenous CoA-independent succinic semialdehyde dehydrogenase, but isdeficient in 4-hydroxybutanoate dehydrogenase, then an encoding nucleicacid is needed for this enzyme to achieve 4-HB biosynthesis. Thus, anon-naturally occurring microbial organism of the invention can beproduced by introducing exogenous enzyme or protein activities to obtaina desired biosynthetic pathway or a desired biosynthetic pathway can beobtained by introducing one or more exogenous enzyme or proteinactivities that, together with one or more endogenous enzymes orproteins, produces a desired product such as 4-HB or BDO.

In like fashion, where 4-HB biosynthesis is selected to occur throughthe succinate to succinyl-CoA pathway (the succinyl-CoA pathway),encoding nucleic acids for host deficiencies in the enzymes succinyl-CoAsynthetase, CoA-dependent succinic semialdehyde dehydrogenase and/or4-hydroxybutanoate dehydrogenase are to be exogenously expressed in therecipient host. Selection of 4-HB biosynthesis through theα-ketoglutarate to succinic semialdehyde pathway (the α-ketoglutaratepathway) can utilize exogenous expression for host deficiencies in oneor more of the enzymes for glutamate:succinic semialdehyde transaminase,glutamate decarboxylase and/or 4-hydroxybutanoate dehydrogenase, orα-ketoglutarate decarboxylase and 4-hydroxybutanoate dehydrogenase. Oneskilled in the art can readily determine pathway enzymes for productionof 4-HB or BDO, as disclosed herein.

Depending on the 4-HB or BDO biosynthetic pathway constituents of aselected host microbial organism, the non-naturally occurring microbialorganisms of the invention will include at least one exogenouslyexpressed 4-HB or BDO pathway-encoding nucleic acid and up to allencoding nucleic acids for one or more 4-HB or BDO biosyntheticpathways. For example, 4-HB or BDO biosynthesis can be established in ahost deficient in a pathway enzyme or protein through exogenousexpression of the corresponding encoding nucleic acid. In a hostdeficient in all enzymes or proteins of a 4-HB or BDO pathway, exogenousexpression of all enzyme or proteins in the pathway can be included,although it is understood that all enzymes or proteins of a pathway canbe expressed even if the host contains at least one of the pathwayenzymes or proteins. For example, exogenous expression of all enzymes orproteins in a pathway for production of BDO can be included. Forexample, 4-HB biosynthesis can be established from all five pathways ina host deficient in 4-hydroxybutanoate dehydrogenase through exogenousexpression of a 4-hydroxybutanoate dehydrogenase encoding nucleic acid.In contrast, 4-HB biosynthesis can be established from all five pathwaysin a host deficient in all eight enzymes through exogenous expression ofall eight of CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate: succinic semialdehyde transaminase, glutamatedecarboxylase, α-ketoglutarate decarboxylase, α-ketoglutaratedehydrogenase and 4-hydroxybutanoate dehydrogenase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the 4-HB orBDO pathway deficiencies of the selected host microbial organism.Therefore, a non-naturally occurring microbial organism of the inventioncan have one, two, three, four, five, six, seven, eight or up to allnucleic acids encoding the enzymes disclosed herein constituting one ormore 4-HB or BDO biosynthetic pathways. In some embodiments, thenon-naturally occurring microbial organisms also can include othergenetic modifications that facilitate or optimize 4-HB or BDObiosynthesis or that confer other useful functions onto the hostmicrobial organism. One such other functionality can include, forexample, augmentation of the synthesis of one or more of the 4-HBpathway precursors such as succinate, succinyl-CoA, α-ketoglutarate,4-aminobutyrate, glutamate, acetoacetyl-CoA, and/or homoserine.

Generally, a host microbial organism is selected such that it producesthe precursor of a 4-HB or BDO pathway, either as a naturally producedmolecule or as an engineered product that either provides de novoproduction of a desired precursor or increased production of a precursornaturally produced by the host microbial organism. For example,succinyl-CoA, α-ketoglutarate, 4-aminobutyrate, glutamate,acetoacetyl-CoA, and homoserine are produced naturally in a hostorganism such as E. coli. A host organism can be engineered to increaseproduction of a precursor, as disclosed herein. In addition, a microbialorganism that has been engineered to produce a desired precursor can beused as a host organism and further engineered to express enzymes orproteins of a 4-HB or BDO pathway.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize 4-HB or BDO. In this specific embodiment it canbe useful to increase the synthesis or accumulation of a 4-HB or BDOpathway product to, for example, drive 4-HB or BDO pathway reactionstoward 4-HB or BDO production. Increased synthesis or accumulation canbe accomplished by, for example, overexpression of nucleic acidsencoding one or more of the 4-HB or BDO pathway enzymes disclosedherein. Over expression of the 4-HB or BDO pathway enzyme or enzymes canoccur, for example, through exogenous expression of the endogenous geneor genes, or through exogenous expression of the heterologous gene orgenes. Therefore, naturally occurring organisms can be readily generatedto be non-naturally occurring 4-HB or BDO producing microbial organismsof the invention through overexpression of one, two, three, four, five,six and so forth up to all nucleic acids encoding 4-HB or BDObiosynthetic pathway enzymes. In addition, a non-naturally occurringorganism can be generated by mutagenesis of an endogenous gene thatresults in an increase in activity of an enzyme in the 4-HB or BDObiosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism (see Examples).

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

Sources of encoding nucleic acids for a 4-HB or BDO pathway enzyme caninclude, for example, any species where the encoded gene product iscapable of catalyzing the referenced reaction. Such species include bothprokaryotic and eukaryotic organisms including, but not limited to,bacteria, including archaea and eubacteria, and eukaryotes, includingyeast, plant, insect, animal, and mammal, including human. Exemplaryspecies for such sources include, for example, those organisms listedbelow as well as other exemplary species disclosed herein or availableas source organisms for corresponding genes, including but not limitedto Escherichia coli, Saccharomyces cerevisiae, Saccharomyces kluyveri,Clostridium kluyveri, Clostridium acetobutylicum, Clostridiumbeijerinckii, Clostridium saccharoperbutylacetonicum, Clostridiumperfringens, Clostridium difficile, Clostridium botulinum, Clostridiumtyrobutyricum, Clostridium tetanomorphum, Clostridium tetani,Clostridium propionicum, Clostridium aminobutyricum, Clostridiumsubterminale, Clostridium sticklandii, Ralstonia eutropha, Mycobacteriumbovis, Mycobacterium tuberculosis, Porphyromonas gingivalis, Arabidopsisthaliana, Thermus thermophilus, Pseudomonas species, includingPseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri,Pseudomonas fluorescens, Homo sapiens, Oryctolagus cuniculus,Rhodobacter spaeroides, Thermoanaerobacter brockii, Metallosphaerasedula, Leuconostoc mesenteroides, Chloroflexus aurantiacus, Roseiflexuscastenholzii, Erythrobacter, Simmondsia chinensis, Acinetobacterspecies, including Acinetobacter calcoaceticus and Acinetobacter baylyi,Porphyromonas gingivalis, Sulfolobus tokodaii, Sulfolobus solfataricus,Sulfolobus acidocaldarius, Bacillus subtilis, Bacillus cereus, Bacillusmegaterium, Bacillus brevis, Bacillus pumilus, Rattus norvegicus,Klebsiella pneumonia, Klebsiella oxytoca, Euglena gracilis, Treponemadenticola, Moorella thermoacetica, Thermotoga maritima, Halobacteriumsalinarum, Geobacillus stearothermophilus, Aeropyrum pernix, Sus scrofa,Caenorhabditis elegans, Corynebacterium glutamicum, Acidaminococcusfermentans, Lactococcus lactis, Lactobacillus plantarum, Streptococcusthermophilus, Enterobacter aerogenes, Candida, Aspergillus terreus,Pedicoccus pentosaceus, Zymomonas mobilus, Acetobacter pasteurians,Kluyveromyces lactis, Eubacterium barkeri, Bacteroides capillosus,Anaerotruncus colihominis, Natranaerobius thermophilusm, Campylobacterjejuni, Haemophilus influenzae, Serratia marcescens, Citrobacteramalonaticus, Myxococcus xanthus, Fusobacterium nuleatum, Penicilliumchrysogenum marine gamma proteobacterium, butyrate-producing bacterium,and others disclosed herein (see Examples). For example, microbialorganisms having 4-HB or BDO biosynthetic production are exemplifiedherein with reference to E. coli and yeast hosts. However, with thecomplete genome sequence available for now more than 550 species (withmore than half of these available on public databases such as the NCBI),including 395 microorganism genomes and a variety of yeast, fungi,plant, and mammalian genomes, the identification of genes encoding therequisite 4-HB or BDO biosynthetic activity for one or more genes inrelated or distant species, including for example, homologues,orthologs, paralogs and nonorthologous gene displacements of knowngenes, and the interchange of genetic alterations between organisms isroutine and well known in the art. Accordingly, the metabolicalterations enabling biosynthesis of 4-HB or BDO and other compounds ofthe invention described herein with reference to a particular organismsuch as E. coli or yeast can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative 4-HB or BDO biosyntheticpathway exists in an unrelated species, 4-HB or BDO biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual genes usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesize 4-HB,such as monomeric 4-HB, or BDO.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger andPichia pastoris. E. coli is a particularly useful host organisms sinceit is a well characterized microbial organism suitable for geneticengineering. Other particularly useful host organisms include yeast suchas Saccharomyces cerevisiae.

Methods for constructing and testing the expression levels of anon-naturally occurring 4-HB- or BDO-producing host can be performed,for example, by recombinant and detection methods well known in the art.Such methods can be found described in, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring HarborLaboratory, New York (2001); Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1999). 4-HB andGBL can be separated by, for example, HPLC using a Spherisorb 5 ODS1column and a mobile phase of 70% 10 mM phosphate buffer (pH=7) and 30%methanol, and detected using a UV detector at 215 nm (Hennessy et al.2004, J. Forensic Sci. 46(6):1-9). BDO is detected by gas chromatographyor by HPLC and refractive index detector using an Aminex HPX-87H columnand a mobile phase of 0.5 mM sulfuric acid (Gonzalez-Pajuelo et al.,Met. Eng. 7:329-336 (2005)).

Exogenous nucleic acid sequences involved in a pathway for production of4-HB or BDO can be introduced stably or transiently into a host cellusing techniques well known in the art including, but not limited to,conjugation, electroporation, chemical transformation, transduction,transfection, and ultrasound transformation. For exogenous expression inE. coli or other prokaryotic cells, some nucleic acid sequences in thegenes or cDNAs of eukaryotic nucleic acids can encode targeting signalssuch as an N-terminal mitochondrial or other targeting signal, which canbe removed before transformation into prokaryotic host cells, ifdesired. For example, removal of a mitochondrial leader sequence led toincreased expression in E. coli (Hoffmeister et al., J. Biol. Chem.280:4329-4338 (2005)). For exogenous expression in yeast or othereukaryotic cells, genes can be expressed in the cytosol without theaddition of leader sequence, or can be targeted to mitochondrion orother organelles, or targeted for secretion, by the addition of asuitable targeting sequence such as a mitochondrial targeting orsecretion signal suitable for the host cells. Thus, it is understoodthat appropriate modifications to a nucleic acid sequence to remove orinclude a targeting sequence can be incorporated into an exogenousnucleic acid sequence to impart desirable properties. Furthermore, genescan be subjected to codon optimization with techniques well known in theart to achieve optimized expression of the proteins.

An expression vector or vectors can be constructed to harbor one or more4-HB biosynthetic pathway and/or one or more BDO biosynthetic encodingnucleic acids as exemplified herein operably linked to expressioncontrol sequences functional in the host organism. Expression vectorsapplicable for use in the microbial host organisms of the inventioninclude, for example, plasmids, phage vectors, viral vectors, episomesand artificial chromosomes, including vectors and selection sequences ormarkers operable for stable integration into a host chromosome.Additionally, the expression vectors can include one or more selectablemarker genes and appropriate expression control sequences. Selectablemarker genes also can be included that, for example, provide resistanceto antibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive and inducible promoters,transcription enhancers, transcription terminators, and the like whichare well known in the art. When two or more exogenous encoding nucleicacids are to be co-expressed, both nucleic acids can be inserted, forexample, into a single expression vector or in separate expressionvectors. For single vector expression, the encoding nucleic acids can beoperationally linked to one common expression control sequence or linkedto different expression control sequences, such as one induciblepromoter and one constitutive promoter. The transformation of exogenousnucleic acid sequences involved in a metabolic or synthetic pathway canbe confirmed using methods well known in the art. Such methods include,for example, nucleic acid analysis such as Northern blots or polymerasechain reaction (PCR) amplification of mRNA, or immunoblotting forexpression of gene products, or other suitable analytical methods totest the expression of an introduced nucleic acid sequence or itscorresponding gene product. It is understood by those skilled in the artthat the exogenous nucleic acid is expressed in a sufficient amount toproduce the desired product, and it is further understood thatexpression levels can be optimized to obtain sufficient expression usingmethods well known in the art and as disclosed herein.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding a 4-HB or BDOpathway enzyme in sufficient amounts to produce 4-HB, such as monomeric4-HB, or BDO. It is understood that the microbial organisms of theinvention are cultured under conditions sufficient to produce 4-HB orBDO. Exemplary levels of expression for 4-HB enzymes in each pathway aredescribed further below in the Examples. Following the teachings andguidance provided herein, the non-naturally occurring microbialorganisms of the invention can achieve biosynthesis of 4-HB, such asmonomeric 4-HB, or BDO resulting in intracellular concentrations betweenabout 0.1-200 mM or more, for example, 0.1-25 mM or more. Generally, theintracellular concentration of 4-HB, such as monomeric 4-HB, or BDO isbetween about 3-150 mM or more, particularly about 5-125 mM or more, andmore particularly between about 8-100 mM, for example, about 3-20 mM,particularly between about 5-15 mM and more particularly between about8-12 mM, including about 10 mM, 20 mM, 50 mM, 80 mM or more.Intracellular concentrations between and above each of these exemplaryranges also can be achieved from the non-naturally occurring microbialorganisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007. Any of theseconditions can be employed with the non-naturally occurring microbialorganisms as well as other anaerobic conditions well known in the art.Under such anaerobic conditions, the 4-HB or BDO producers cansynthesize 4-HB or BDO at intracellular concentrations of 5-10 mM ormore as well as all other concentrations exemplified herein. It isunderstood that, even though the above description refers tointracellular concentrations, 4-HB or BDO producing microbial organismscan produce 4-HB or BDO intracellularly and/or secrete the product intothe culture medium.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of 4-HB or BDO includes anaerobic culture or fermentationconditions. In certain embodiments, the non-naturally occurringmicrobial organisms of the invention can be sustained, cultured orfermented under anaerobic or substantially anaerobic conditions.Briefly, anaerobic conditions refers to an environment devoid of oxygen.Substantially anaerobic conditions include, for example, a culture,batch fermentation or continuous fermentation such that the dissolvedoxygen concentration in the medium remains between 0 and 10% ofsaturation. Substantially anaerobic conditions also includes growing orresting cells in liquid medium or on solid agar inside a sealed chambermaintained with an atmosphere of less than 1% oxygen. The percent ofoxygen can be maintained by, for example, sparging the culture with anN₂/CO₂ mixture or other suitable non-oxygen gas or gases.

The invention also provides a non-naturally occurring microbialbiocatalyst including a microbial organism having 4-hydroxybutanoic acid(4-HB) and 1,4-butanediol (BDO) biosynthetic pathways that include atleast one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, 4-hydroxybutyrate:CoA transferase, glutamate: succinicsemialdehyde transaminase, glutamate decarboxylase, CoA-independentaldehyde dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcoholdehydrogenase, wherein the exogenous nucleic acid is expressed insufficient amounts to produce 1,4-butanediol (BDO).4-Hydroxybutyrate:CoA transferase also is known as 4-hydroxybutyrylCoA:acetyl-CoA transferase. Additional 4-HB or BDO pathway enzymes arealso disclosed herein (see Examples and FIGS. 8-13).

The invention further provides non-naturally occurring microbialbiocatalyst including a microbial organism having 4-hydroxybutanoic acid(4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathwaysinclude at least one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, succinyl-CoA synthetase, CoA-dependent succinicsemialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase,4-butyrate kinase, phosphotransbutyrylase, α-ketoglutaratedecarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or analdehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid isexpressed in sufficient amounts to produce 1,4-butanediol (BDO).

Non-naturally occurring microbial organisms also can be generated whichbiosynthesize BDO. As with the 4-HB producing microbial organisms of theinvention, the BDO producing microbial organisms also can produceintracellularly or secret the BDO into the culture medium. Following theteachings and guidance provided previously for the construction ofmicrobial organisms that synthesize 4-HB, additional BDO pathways can beincorporated into the 4-HB producing microbial organisms to generateorganisms that also synthesize BDO and other BDO family compounds. Thechemical synthesis of BDO and its downstream products are known. Thenon-naturally occurring microbial organisms of the invention capable ofBDO biosynthesis circumvent these chemical synthesis using 4-HB as anentry point as illustrated in FIG. 1. As described further below, the4-HB producers also can be used to chemically convert 4-HB to GBL andthen to BDO or THF, for example. Alternatively, the 4-HB producers canbe further modified to include biosynthetic capabilities for conversionof 4-HB and/or GBL to BDO.

The additional BDO pathways to introduce into 4-HB producers include,for example, the exogenous expression in a host deficient background orthe overexpression of one or more of the enzymes exemplified in FIG. 1as steps 9-13. One such pathway includes, for example, the enzymeactivities necessary to carryout the reactions shown as steps 9, 12 and13 in FIG. 1, where the aldehyde and alcohol dehydrogenases can beseparate enzymes or a multifunctional enzyme having both aldehyde andalcohol dehydrogenase activity. Another such pathway includes, forexample, the enzyme activities necessary to carry out the reactionsshown as steps 10, 11, 12 and 13 in FIG. 1, also where the aldehyde andalcohol dehydrogenases can be separate enzymes or a multifunctionalenzyme having both aldehyde and alcohol dehydrogenase activity.Accordingly, the additional BDO pathways to introduce into 4-HBproducers include, for example, the exogenous expression in a hostdeficient background or the overexpression of one or more of a4-hydroxybutyrate:CoA transferase, butyrate kinase,phosphotransbutyrylase, CoA-independent aldehyde dehydrogenase,CoA-dependent aldehyde dehydrogenase or an alcohol dehydrogenase. In theabsence of endogenous acyl-CoA synthetase capable of modifying 4-HB, thenon-naturally occurring BDO producing microbial organisms can furtherinclude an exogenous acyl-CoA synthetase selective for 4-HB, or thecombination of multiple enzymes that have as a net reaction conversionof 4-HB into 4-HB-CoA. As exemplified further below in the Examples,butyrate kinase and phosphotransbutyrylase exhibit BDO pathway activityand catalyze the conversions illustrated in FIG. 1 with a 4-HBsubstrate. Therefore, these enzymes also can be referred to herein as4-hydroxybutyrate kinase and phosphotranshydroxybutyrylase respectively.

Exemplary alcohol and aldehyde dehydrogenases that can be used for thesein vivo conversions from 4-HB to BDO are listed below in Table 1.

TABLE 1 Alcohol and Aldehyde Dehydrogenases for Conversion of 4-HB toBDO. ALCOHOL DEHYDROGENASES ec: 1.1.1.1 alcohol dehydrogenase ec:1.1.1.2 alcohol dehydrogenase (NADP+) ec: 1.1.1.4 (R,R)-butanedioldehydrogenase ec: 1.1.1.5 acetoin dehydrogenase ec: 1.1.1.6 glyceroldehydrogenase ec: 1.1.1.7 propanediol-phosphate dehydrogenase ec:1.1.1.8 glycerol-3-phosphate dehydrogenase (NAD+) ec: 1.1.1.11D-arabinitol 4-dehydrogenase ec: 1.1.1.12 L-arabinitol 4-dehydrogenaseec: 1.1.1.13 L-arabinitol 2-dehydrogenase ec: 1.1.1.14 L-iditol2-dehydrogenase ec: 1.1.1.15 D-iditol 2-dehydrogenase ec: 1.1.1.16galactitol 2-dehydrogenase ec: 1.1.1.17 mannitol-1-phosphate 5-dehydrogenase ec: 1.1.1.18 inositol 2-dehydrogenase ec: 1.1.1.21aldehyde reductase ec: 1.1.1.23 histidinol dehydrogenase ec: 1.1.1.26glyoxylate reductase ec: 1.1.1.27 L-lactate dehydrogenase ec: 1.1.1.28D-lactate dehydrogenase ec: 1.1.1.29 glycerate dehydrogenase ec:1.1.1.30 3-hydroxybutyrate dehydrogenase ec: 1.1.1.313-hydroxyisobutyrate dehydrogenase ec: 1.1.1.35 3-hydroxyacyl-CoAdehydrogenase ec: 1.1.1.36 acetoacetyl-CoA reductase ec: 1.1.1.37 malatedehydrogenase ec: 1.1.1.38 malate dehydrogenase(oxaloacetate-decarboxylating) ec: 1.1.1.39 malate dehydrogenase(decarboxylating) ec: 1.1.1.40 malate dehydrogenase(oxaloacetate-decarboxylating) (NADP+) ec: 1.1.1.41 isocitratedehydrogenase (NAD+) ec: 1.1.1.42 isocitrate dehydrogenase (NADP+) ec:1.1.1.54 allyl-alcohol dehydrogenase ec: 1.1.1.55 lactaldehyde reductase(NADPH) ec: 1.1.1.56 ribitol 2-dehydrogenase ec: 1.1.1.593-hydroxypropionate dehydrogenase ec: 1.1.1.60 2-hydroxy-3-oxopropionatereductase ec: 1.1.1.61 4-hydroxybutyrate dehydrogenase ec: 1.1.1.66omega-hydroxydecanoate dehydrogenase ec: 1.1.1.67 mannitol2-dehydrogenase ec: 1.1.1.71 alcohol dehydrogenase [NAD(P)+] ec:1.1.1.72 glycerol dehydrogenase (NADP+) ec: 1.1.1.73 octanoldehydrogenase ec: 1.1.1.75 (R)-aminopropanol dehydrogenase ec: 1.1.1.76(S,S)-butanediol dehydrogenase ec: 1.1.1.77 lactaldehyde reductase ec:1.1.1.78 methylglyoxal reductase (NADH- dependent) ec: 1.1.1.79glyoxylate reductase (NADP+) ec: 1.1.1.80 isopropanol dehydrogenase(NADP+) ec: 1.1.1.81 hydroxypyruvate reductase ec: 1.1.1.82 malatedehydrogenase (NADP+) ec: 1.1.1.83 D-malate dehydrogenase(decarboxylating) ec: 1.1.1.84 dimethylmalate dehydrogenase ec: 1.1.1.853-isopropylmalate dehydrogenase ec: 1.1.1.86 ketol-acid reductoisomeraseec: 1.1.1.87 homoisocitrate dehydrogenase ec: 1.1.1.88hydroxymethylglutaryl-CoA reductase ec: 1.1.1.90 aryl-alcoholdehydrogenase ec: 1.1.1.91 aryl-alcohol dehydrogenase (NADP+) ec:1.1.1.92 oxaloglycolate reductase (decarboxylating) ec: 1.1.1.94glycerol-3-phosphate dehydrogenase [NAD(P)+] ec: 1.1.1.95phosphoglycerate dehydrogenase ec: 1.1.1.97 3-hydroxybenzyl-alcoholdehydrogenase ec: 1.1.1.101 acylglycerone-phosphate reductase ec:1.1.1.103 L-threonine 3-dehydrogenase ec: 1.1.1.104 4-oxoprolinereductase ec: 1.1.1.105 retinol dehydrogenase ec: 1.1.1.110indolelactate dehydrogenase ec: 1.1.1.112 indanol dehydrogenase ec:1.1.1.113 L-xylose 1-dehydrogenase ec: 1.1.1.129 L-threonate3-dehydrogenase ec: 1.1.1.137 ribitol-5-phosphate 2- dehydrogenase ec:1.1.1.138 mannitol 2-dehydrogenase (NADP+) ec: 1.1.1.140sorbitol-6-phosphate 2- dehydrogenase ec: 1.1.1.142 D-pinitoldehydrogenase ec: 1.1.1.143 sequoyitol dehydrogenase ec: 1.1.1.144perillyl-alcohol dehydrogenase ec: 1.1.1.156 glycerol 2-dehydrogenase(NADP+) ec: 1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase ec: 1.1.1.163cyclopentanol dehydrogenase ec: 1.1.1.164 hexadecanol dehydrogenase ec:1.1.1.165 2-alkyn-1-ol dehydrogenase ec: 1.1.1.166hydroxycyclohexanecarboxylate dehydrogenase ec: 1.1.1.167hydroxymalonate dehydrogenase ec: 1.1.1.174 cyclohexane-1,2-dioldehydrogenase ec: 1.1.1.177 glycerol-3-phosphate 1- dehydrogenase(NADP+) ec: 1.1.1.178 3-hydroxy-2-methylbutyryl-CoA dehydrogenase ec:1.1.1.185 L-glycol dehydrogenase ec: 1.1.1.190 indole-3-acetaldehydereductase (NADH) ec: 1.1.1.191 indole-3-acetaldehyde reductase (NADPH)ec: 1.1.1.192 long-chain-alcohol dehydrogenase ec: 1.1.1.194coniferyl-alcohol dehydrogenase ec: 1.1.1.195 cinnamyl-alcoholdehydrogenase ec: 1.1.1.198 (+)-borneol dehydrogenase ec: 1.1.1.2021,3-propanediol dehydrogenase ec: 1.1.1.207 (−)-menthol dehydrogenaseec: 1.1.1.208 (+)-neomenthol dehydrogenase ec: 1.1.1.216 farnesoldehydrogenase ec: 1.1.1.217 benzyl-2-methyl-hydroxybutyratedehydrogenase ec: 1.1.1.222 (R)-4-hydroxyphenyllactate dehydrogenase ec:1.1.1.223 isopiperitenol dehydrogenase ec: 1.1.1.2264-hydroxycyclohexanecarboxylate dehydrogenase ec: 1.1.1.229 diethyl2-methyl-3-oxosuccinate reductase ec: 1.1.1.237 hydroxyphenylpyruvatereductase ec: 1.1.1.244 methanol dehydrogenase ec: 1.1.1.245cyclohexanol dehydrogenase ec: 1.1.1.250 D-arabinitol 2-dehydrogenaseec: 1.1.1.251 galactitol 1-phosphate 5- dehydrogenase ec: 1.1.1.255mannitol dehydrogenase ec: 1.1.1.256 fluoren-9-ol dehydrogenase ec:1.1.1.257 4- (hydroxymethyl)benzenesulfonate dehydrogenase ec: 1.1.1.2586-hydroxyhexanoate dehydrogenase ec: 1.1.1.259 3-hydroxypimeloyl-CoAdehydrogenase ec :1.1.1.261 glycerol-1-phosphate dehydrogenase [NAD(P)+]ec: 1.1.1.265 3-methylbutanal reductase ec: 1.1.1.283 methylglyoxalreductase (NADPH- dependent) ec: 1.1.1.286 isocitrate-homoisocitratedehydrogenase ec: 1.1.1.287 D-arabinitol dehydrogenase (NADP+) butanoldehydrogenase ALDEHYDE DEHYDROGENASES ec: 1.2.1.2 formate dehydrogenaseec: 1.2.1.3 aldehyde dehydrogenase (NAD+) ec: 1.2.1.4 aldehydedehydrogenase (NADP+) ec: 1.2.1.5 aldehyde dehydrogenase [NAD(P)+] ec:1.2.1.7 benzaldehyde dehydrogenase (NADP+) ec: 1.2.1.8 betaine-aldehydedehydrogenase ec: 1.2.1.9 glyceraldehyde-3-phosphate dehydrogenase(NADP+) ec: 1.2.1.10 acetaldehyde dehydrogenase (acetylating) ec:1.2.1.11 aspartate-semialdehyde dehydrogenase ec: 1.2.1.12glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) ec: 1.2.1.13glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating) ec:1.2.1.15 malonate-semialdehyde dehydrogenase ec: 1.2.1.16succinate-semialdehyde dehydrogenase [NAD(P)+] ec: 1.2.1.17 glyoxylatedehydrogenase (acylating) ec: 1.2.1.18 malonate-semialdehydedehydrogenase (acetylating) ec: 1.2.1.19 aminobutyraldehydedehydrogenase ec: 1.2.1.20 glutarate-semialdehyde dehydrogenase ec:1.2.1.21 glycolaldehyde dehydrogenase ec: 1.2.1.22 lactaldehydedehydrogenase ec: 1.2.1.23 2-oxoaldehyde dehydrogenase (NAD+) ec:1.2.1.24 succinate-semialdehyde dehydrogenase ec: 1.2.1.252-oxoisovalerate dehydrogenase (acylating) ec: 1.2.1.262,5-dioxovalerate dehydrogenase ec: 1.2.1.27 methylmalonate-semialdehydedehydrogenase (acylating) ec: 1.2.1.28 benzaldehyde dehydrogenase (NAD+)ec: 1.2.1.29 aryl-aldehyde dehydrogenase ec: 1.2.1.30 aryl-aldehydedehydrogenase (NADP+) ec: 1.2.1.31 L-aminoadipate-semialdehydedehydrogenase ec: 1.2.1.32 aminomuconate-semialdehyde dehydrogenase ec:1.2.1.36 retinal dehydrogenase ec: 1.2.1.39 phenylacetaldehydedehydrogenase ec: 1.2.1.41 glutamate-5-semialdehyde dehydrogenase ec:1.2.1.42 hexadecanal dehydrogenase (acylating) ec: 1.2.1.43 formatedehydrogenase (NADP+) ec: 1.2.1.45 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase ec: 1.2.1.46 formaldehyde dehydrogenase ec:1.2.1.47 4-trimethylammoniobutyraldehyde dehydrogenase ec: 1.2.1.48long-chain-aldehyde dehydrogenase ec: 1.2.1.49 2-oxoaldehydedehydrogenase (NADP+) ec: 1.2.1.51 pyruvate dehydrogenase (NADP+) ec:1.2.1.52 oxoglutarate dehydrogenase (NADP+) ec: 1.2.1.534-hydroxyphenylacetaldehyde dehydrogenase ec: 1.2.1.57 butanaldehydrogenase ec: 1.2.1.58 phenylglyoxylate dehydrogenase (acylating)ec: 1.2.1.59 glyceraldehyde-3-phosphate dehydrogenase (NAD(P)+)(phosphorylating) ec: 1.2.1.62 4-formylbenzenesulfonate dehydrogenaseec: 1.2.1.63 6-oxohexanoate dehydrogenase ec: 1.2.1.644-hydroxybenzaldehyde dehydrogenase ec: 1.2.1.65 salicylaldehydedehydrogenase ec: 1.2.1.66 mycothiol-dependent formaldehydedehydrogenase ec: 1.2.1.67 vanillin dehydrogenase ec: 1.2.1.68coniferyl-aldehyde dehydrogenase ec: 1.2.1.69 fluoroacetaldehydedehydrogenase ec: 1.2.1.71 succinylglutamate-semialdehyde dehydrogenase

Other exemplary enzymes and pathways are disclosed herein (seeExamples). Furthermore, it is understood that enzymes can be utilizedfor carry out reactions for which the substrate is not the naturalsubstrate. While the activity for the non-natural substrate may be lowerthan the natural substrate, it is understood that such enzymes can beutilized, either as naturally occurring or modified using the directedevolution or adaptive evolution, as disclosed herein (see alsoExamples).

BDO production through any of the pathways disclosed herein are based,in part, on the identification of the appropriate enzymes for conversionof precursors to BDO. A number of specific enzymes for several of thereaction steps have been identified. For those transformations whereenzymes specific to the reaction precursors have not been identified,enzyme candidates have been identified that are best suited forcatalyzing the reaction steps. Enzymes have been shown to operate on abroad range of substrates, as discussed below. In addition, advances inthe field of protein engineering also make it feasible to alter enzymesto act efficiently on substrates, even if not a natural substrate.Described below are several examples of broad-specificity enzymes fromdiverse classes suitable for a BDO pathway as well as methods that havebeen used for evolving enzymes to act on non-natural substrates.

A key class of enzymes in BDO pathways is the oxidoreductases thatinterconvert ketones or aldehydes to alcohols (1.1.1). Numerousexemplary enzymes in this class can operate on a wide range ofsubstrates. An alcohol dehydrogenase (1.1.1.1) purified from the soilbacterium Brevibacterium sp KU 1309 (Hirano et al., J. Biosc. Bioeng.100:318-322 (2005)) was shown to operate on a plethora of aliphatic aswell as aromatic alcohols with high activities. Table 2 shows theactivity of the enzyme and its K_(m) on different alcohols. The enzymeis reversible and has very high activity on several aldehydes also(Table 3).

TABLE 2 Relative activities of an alcohol dehydrogenase fromBrevibacterium sp KU to oxidize various alcohols. Relative ActivityK_(m) Substrate (0%) (mM) 2-Phenylethanol 100* 0.025(S)-2-Phenylpropanol 156 0.157 (R)-2-Phenylpropanol 63 0.020 Bynzylalcohol 199 0.012 3-Phenylpropanol 135 0.033 Ethanol 76 1-Butanol 1111-Octanol 101 1-Dodecanol 68 1-Phenylethanol 46 2-Propanol 54 *Theactivity of 2-phenylethanol, corresponding to 19.2 U/mg, was taken as100%.

TABLE 3 Relative activities of an alcohol dehydrogenase fromBrevibacterium sp KU 1309 to reduce various carbonyl compounds. RelativeActivity K_(m) Substrate (%) (mM) Phenylacetaldehyde 100 0.2612-Phenylpropionaldehyde 188 0.864 1-Octylaldehyde 87 Acetophenone 0

Lactate dehydrogenase (1.1.1.27) from Ralstonia eutropha is anotherenzyme that has been demonstrated to have high activities on several2-oxoacids such as 2-oxobutyrate, 2-oxopentanoate and 2-oxoglutarate (aC5 compound analogous to 2-oxoadipate) (Steinbuchel and Schlegel, Eur.J. Biochem. 130:329-334 (1983)). Column 2 in Table 4 demonstrates theactivities of ldhA from R. eutropha (formerly A. eutrophus) on differentsubstrates (Steinbuchel and Schlegel, supra, 1983).

TABLE 4 The in vitro activity of R. eutropha ldhA (Steinbuchel andSchlegel, supra, 1983) on differentsubstrates and compared with that onpyruvate. L(+)-lactate Activity (%) of D(−)-lactate dehydrogenaseL(+)-lactate dehydrogenase from dehydrogenase from from Substrate A.eutrophus rabbit muscle L. leichmanii Glyoxylate 8.7 23.9 5.0 Pyruvate100.0 100.0 100.0 2-Oxobutyrate 107.0 18.6 1.1 2-Oxovalerate 125.0 0.70.0 3-Methyl-2- 28.5 0.0 0.0 oxobutyrate 3-Methyl-2- 5.3 0.0 0.0oxovalerate 4-Methyl-2- 39.0 1.4 1.1 oxopentanoate Oxaloacetate 0.0 33.123.1 2-Oxoglutarate 79.6 0.0 0.0 3-Fluoropyruvate 33.6 74.3 40.0

Oxidoreductases that can convert 2-oxoacids to their acyl-CoAcounterparts (1.2.1) have been shown to accept multiple substrates aswell. For example, branched-chain 2-keto-acid dehydrogenase complex(BCKAD), also known as 2-oxoisovalerate dehydrogenase (1.2.1.25),participates in branched-chain amino acid degradation pathways,converting 2-keto acids derivatives of valine, leucine and isoleucine totheir acyl-CoA derivatives and CO₂. In some organisms including Rattusnorvegicus (Paxton et al., Biochem. J. 234:295-303 (1986)) andSaccharomyces cerevisiae (Sinclair et al., Biochem. Mol Biol. Int.32:911-922 (1993), this complex has been shown to have a broad substraterange that includes linear oxo-acids such as 2-oxobutanoate andalpha-ketoglutarate, in addition to the branched-chain amino acidprecursors.

Members of yet another class of enzymes, namely aminotransferases(2.6.1), have been reported to act on multiple substrates. Aspartateaminotransferase (aspAT) from Pyrococcus fursious has been identified,expressed in E. coli and the recombinant protein characterized todemonstrate that the enzyme has the highest activities towards aspartateand alpha-ketoglutarate but lower, yet significant activities towardsalanine, glutamate and the aromatic amino acids (Ward et al., Archaea133-141 (2002)). In another instance, an aminotransferase identifiedfrom Leishmania mexicana and expressed in E. coli (Vernal et al., FEMSMicrobiol. Lett. 229:217-222 (2003)) was reported to have a broadsubstrate specificity towards tyrosine (activity considered 100% ontyrosine), phenylalanine (90%), tryptophan (85%), aspartate (30%),leucine (25%) and methionine (25%), respectively (Vernal et al., Mol.Biochem. Parasitol 96:83-92 (1998)). Similar broad specificity has beenreported for a tyrosine aminotransferase from Trypanosoma cruzi, eventhough both of these enzymes have a sequence homology of only 6%. Thelatter enzyme can accept leucine, methionine as well as tyrosine,phenylalanine, tryptophan and alanine as efficient amino donors (Nowickiet al., Biochim. Biophys. Acta 1546: 268-281 (2001)).

CoA transferases (2.8.3) have been demonstrated to have the ability toact on more than one substrate. Specifically, a CoA transferase waspurified from Clostridium acetobutylicum and was reported to have thehighest activities on acetate, propionate, and butyrate. It also hadsignificant activities with valerate, isobutyrate, and crotonate(Wiesenborn et al., Appl. Environ. Microbiol. 55:323-329 (1989)). Inanother study, the E. coli enzyme acyl-CoA:acetate-CoA transferase, alsoknown as acetate-CoA transferase (EC 2.8.3.8), has been shown totransfer the CoA moiety to acetate from a variety of branched and linearacyl-CoA substrates, including isobutyrate (Matthies and Schink, App.Environm. Microbiol. 58:1435-1439 (1992)), valerate (Vanderwinkel etal., Biochem. Biophys. Res Commun. 33:902-908 (1968b)) and butanoate(Vanderwinkel et al., Biochem. Biophys. Res Commun. 33:902-908(1968a).

Other enzyme classes additionally support broad substrate specificityfor enzymes. Some isomerases (5.3.3) have also been proven to operate onmultiple substrates. For example, L-rhamnose isomerase from Pseudomonasstutzeri catalyzes the isomerization between various aldolases andketoses (Yoshida et al., J. Mol. Biol. 365:1505-1516 (2007)). Theseinclude isomerization between L-rhamnose and L-rhamnulose, L-mannose andL-fructose, L-xylose and L-xylulose, D-ribose and D-ribulose, andD-allose and D-psicose.

In yet another class of enzymes, the phosphotransferases (2.7.1), thehomoserine kinase (2.7.1.39) from E. coli that converts L-homoserine toL-homoserine phosphate, was found to phosphorylate numerous homoserineanalogs. In these substrates, the carboxyl functional group at theR-position had been replaced by an ester or by a hydroxymethyl group(Huo and Viola, Biochemistry 35:16180-16185 (1996)). Table 5demonstrates the broad substrate specificity of this kinase.

TABLE 5 The substrate specificity of homoserine kinase. % Substratek_(cat) k_(cat) K_(m) (mM) k_(cat)/K_(m) L-homoserine 18.3 ± 0.1 1000.14 ± 0.04 184 ± 17  D-homoserine  8.3 ± 1.1 32 31.8 ± 7.2  0.26 ± 0.03L-aspartate β-semialdehyde  2.1 ± 0.1 8.2 0.28 ± 0.02 7.5 ± 0.3L-2-amino-1,4-butanediol  2.0 ± 0.5 7.9 11.6 ± 6.5  0.17 ± 0.06L-2-amino-5-  2.5 ± 0.4 9.9 1.1 ± 0.5 2.3 ± 0.3 hydroxyvalerateL-homoserine methyl ester 14.7 ± 2.6 80 4.9 ± 2.0 3.0 ± 0.6 L-homoserineethyl ester 13.6 ± 0.8 74 1.9 ± 0.5 7.2 ± 1.7 L-homoserine isopropyl13.6 ± 1.4 74 1.2 ± 0.5 11.3 ± 1.1  ester L-homoserine n-propyl 14.0 ±0.4 76 3.5 ± 0.4 4.0 ± 1.2 ester L-homoserine isobutyl 16.4 ± 0.8 84 6.9± 1.1 2.4 ± 0.3 ester L-homserine n-butyl ester 29.1 ± 1.2 160 5.8 ± 0.85.0 ± 0.5

Another class of enzymes useful in BDO pathways is the acid-thiolligases (6.2.1). Like enzymes in other classes, certain enzymes in thisclass have been determined to have broad substrate specificity. Forexample, acyl CoA ligase from Pseudomonas putida has been demonstratedto work on several aliphatic substrates including acetic, propionic,butyric, valeric, hexanoic, heptanoic, and octanoic acids and onaromatic compounds such as phenylacetic and phenoxyacetic acids(Fernandez-Valverde et al., Appl. Environ. Microbiol. 59:1149-1154(1993)). A related enzyme, malonyl CoA synthetase (6.3.4.9) fromRhizobium trifolii could convert several diacids, namely, ethyl-,propyl-, allyl-, isopropyl-, dimethyl-, cyclopropyl-,cyclopropylmethylene-, cyclobutyl-, and benzyl-malonate into theircorresponding monothioesters (Pohl et al., J. Am. Chem. Soc.123:5822-5823 (2001)). Similarly, decarboxylases (4.1.1) have also beenfound with broad substrate ranges. Pyruvate decarboxylase (PDC), alsotermed keto-acid decarboxylase, is a key enzyme in alcoholicfermentation, catalyzing the decarboxylation of pyruvate toacetaldehyde. The enzyme isolated from Saccharomyces cerevisiae has abroad substrate range for aliphatic 2-keto acids including2-ketobutyrate, 2-ketovalerate, and 2-phenylpyruvate (Li and Jordan,Biochemistry 38:10004-10012 (1999)). Similarly, benzoylformatedecarboxylase has a broad substrate range and has been the target ofenzyme engineering studies. The enzyme from Pseudomonas putida has beenextensively studied and crystal structures of this enzyme are available(Polovnikova et al., Biochemistry 42:1820-1830 (2003); Hasson et al.,Biochemistry 37:9918-9930 (1998)). Branched chain alpha-ketoaciddecarboxylase (BCKA) has been shown to act on a variety of compoundsvarying in chain length from 3 to 6 carbons (Oku and Kaneda, J. Biol.Chem. 263:18386-18396 (1998); Smit et al., Appl. Environ. Microbiol.71:303-311 (2005)). The enzyme in Lactococcus lactis has beencharacterized on a variety of branched and linear substrates including2-oxobutanoate, 2-oxohexanoate, 2-oxopentanoate,3-methyl-2-oxobutanoate, 4-methyl-2-oxobutanoate and isocaproate (Smitet al., Appl. Environ. Microbiol. 71:303-311 (2005).

Interestingly, enzymes known to have one dominant activity have alsobeen reported to catalyze a very different function. For example, thecofactor-dependent phosphoglycerate mutase (5.4.2.1) from Bacillusstearothermophilus and Bacillus subtilis is known to function as aphosphatase as well (Rigden et al., Protein Sci. 10:1835-1846 (2001)).The enzyme from B. stearothermophilus is known to have activity onseveral substrates, including 3-phosphoglycerate,alpha-napthylphosphate, p-nitrophenylphosphate, AMP,fructose-6-phosphate, ribose-5-phosphate and CMP.

In contrast to these examples where the enzymes naturally have broadsubstrate specificities, numerous enzymes have been modified usingdirected evolution to broaden their specificity towards theirnon-natural substrates. Alternatively, the substrate preference of anenzyme has also been changed using directed evolution. Therefore, it isfeasible to engineer a given enzyme for efficient function on a natural,for example, improved efficiency, or a non-natural substrate, forexample, increased efficiency. For example, it has been reported thatthe enantioselectivity of a lipase from Pseudomonas aeruginosa wasimproved significantly (Reetz et al., Agnew. Chem. Int. Ed Engl.36:2830-2832 (1997)). This enzyme hydrolyzed p-nitrophenyl2-methyldecanoate with only 2% enantiomeric excess (ee) in favor of the(S)-acid. However, after four successive rounds of error-pronemutagenesis and screening, a variant was produced that catalyzed therequisite reaction with 81% ee (Reetz et al., Agnew. Chem. Int. Ed Engl.36:2830-2832 (1997)).

Directed evolution methods have been used to modify an enzyme tofunction on an array of non-natural substrates. The substratespecificity of the lipase in P. aeruginosa was broadened byrandomization of amino acid residues near the active site. This allowedfor the acceptance of alpha-substituted carboxylic acid esters by thisenzyme (Reetz et al., Agnew. Chem. Int. Ed Engl. 44:4192-4196 (2005)).In another successful modification of an enzyme, DNA shuffling wasemployed to create an Escherichia coli aminotransferase that acceptedβ-branched substrates, which were poorly accepted by the wild-typeenzyme (Yano et al., Proc. Nat. Acad. Sci. U.S.A. 95:5511-5515 (1998)).Specifically, at the end of four rounds of shuffling, the activity ofaspartate aminotransferase for valine and 2-oxovaline increased by up tofive orders of magnitude, while decreasing the activity towards thenatural substrate, aspartate, by up to 30-fold. Recently, an algorithmwas used to design a retro-aldolase that could be used to catalyze thecarbon-carbon bond cleavage in a non-natural and non-biologicalsubstrate, 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone (Jiang et al.,Science 319:1387-1391 (2008)). These algorithms used differentcombinations of four different catalytic motifs to design new enzyme,and 20 of the selected designs for experimental characterization hadfour-fold improved rates over the uncatalyzed reaction (Jiang et al.,Science 319:1387-1391 (2008)). Thus, not only are these engineeringapproaches capable of expanding the array of substrates on which anenzyme can act, but they allow the design and construction of veryefficient enzymes. For example, a method of DNA shuffling (randomchimeragenesis on transient templates or RACHITT) was reported to leadto an engineered monooxygenase that had an improved rate ofdesulfurization on complex substrates as well as 20-fold fasterconversion of a non-natural substrate (Coco et al., Nat. Biotechnol.19:354-359 (2001)). Similarly, the specific activity of a sluggishmutant triosephosphate isomerase enzyme was improved up to 19-fold from1.3 fold (Hermes et al., Proc. Nat. Acad. Sci. U.S.A. 87:696-700 1990)).This enhancement in specific activity was accomplished by using randommutagenesis over the whole length of the protein and the improvementcould be traced back to mutations in six amino acid residues.

The effectiveness of protein engineering approaches to alter thesubstrate specificity of an enzyme for a desired substrate has also beendemonstrated in several studies. Isopropylmalate dehydrogenase fromThermus thermophilus was modified by changing residues close to theactive site so that it could now act on malate and D-lactate assubstrates (Fujita et al., Biosci. Biotechnol. Biochem. 65:2695-2700(2001)). In this study as well as in others, it was pointed out that oneor a few residues could be modified to alter the substrate specificity.For example, the dihydroflavonol 4-reductase for which a single aminoacid was changed in the presumed substrate-binding region couldpreferentially reduce dihydrokaempferol (Johnson et al., Plant. J.25:325-333 (2001)). The substrate specificity of a very specificisocitrate dehydrogenase from Escherichia coli was changed formisocitrate to isopropylmalate by changing one residue in the active site(Doyle et al., Biochemistry 40:4234-4241 (2001)). Similarly, thecofactor specificity of a NAD⁺-dependent 1,5-hydroxyprostaglandindehydrogenase was altered to NADP⁺ by changing a few residues near theN-terminal end (Cho et al., Arch. Biochem. Biophys. 419:139-146 (2003)).Sequence analysis and molecular modeling analysis were used to identifythe key residues for modification, which were further studied bysite-directed mutagenesis.

Numerous examples exist spanning diverse classes of enzymes where thefunction of enzyme was changed to favor one non-natural substrate overthe natural substrate of the enzyme. A fucosidase was evolved from agalactosidase in E. coli by DNA shuffling and screening (Zhang et al.,Proc. Natl Acad. Sci. U.S.A. 94:4504-4509 (1997)). Similarly, aspartateaminotransferase from E. coli was converted into a tyrosineaminotransferase using homology modeling and site-directed mutagenesis(Onuffer and Kirsch Protein Sci., 4:1750-1757 (1995)). Site-directedmutagenesis of two residues in the active site of benzoylformatedecarboxylase from P. putida reportedly altered the affinity (K_(m))towards natural and non-natural substrates (Siegert et al., Protein EngDes Sel 18:345-357 (2005)). Cytochrome c peroxidase (CCP) fromSaccharomyces cerevisiae was subjected to directed molecular evolutionto generate mutants with increased activity against the classicalperoxidase substrate guaiacol, thus changing the substrate specificityof CCP from the protein cytochrome c to a small organic molecule. Afterthree rounds of DNA shuffling and screening, mutants were isolated whichpossessed a 300-fold increased activity against guaiacol and up to1000-fold increased specificity for this substrate relative to that forthe natural substrate (Iffland et al., Biochemistry 39:10790-10798(2000)).

In some cases, enzymes with different substrate preferences than eitherof the parent enzymes have been obtained. For example,biphenyl-dioxygenase-mediated degradation of polychlorinated biphenylswas improved by shuffling genes from two bacteria, Pseudomonaspseudoalcaligens and Burkholderia cepacia (Kumamaru et al., Nat.Biotechnol. 16:663-666 (1998)). The resulting chimeric biphenyloxygenases showed different substrate preferences than both the parentalenzymes and enhanced the degradation activity towards related biphenylcompounds and single aromatic ring hydrocarbons such as toluene andbenzene which were originally poor substrates for the enzyme.

In addition to changing enzyme specificity, it is also possible toenhance the activities on substrates for which the enzymes naturallyhave low activities. One study demonstrated that amino acid racemasefrom P. putida that had broad substrate specificity (on lysine,arginine, alanine, serine, methionine, cysteine, leucine and histidineamong others) but low activity towards tryptophan could be improvedsignificantly by random mutagenesis (Kino et al., Appl. Microbiol.Biotechnol. 73:1299-1305 (2007)). Similarly, the active site of thebovine BCKAD was engineered to favor alternate substrate acetyl-CoA(Meng and Chuang, Biochemistry 33:12879-12885 (1994)). An interestingaspect of these approaches is that even if random methods have beenapplied to generate these mutated enzymes with efficacious activities,the exact mutations or structural changes that confer the improvement inactivity can be identified. For example, in the aforementioned study,the mutations that facilitated improved activity on tryptophan wastraced back to two different positions.

Directed evolution has also been used to express proteins that aredifficult to express. For example, by subjecting horseradish peroxidaseto random mutagenesis and gene recombination, mutants were identifiedthat had more than 14-fold higher activity than the wild type (Lin etal., Biotechnol. Prog. 15:467-471 (1999)).

Another example of directed evolution shows the extensive modificationsto which an enzyme can be subjected to achieve a range of desiredfunctions. The enzyme lactate dehydrogenase from Bacillusstearothermophilus was subjected to site-directed mutagenesis, and threeamino acid substitutions were made at sites that were believed todetermine the specificity towards different hydroxyacids (Clarke et al.,Biochem. Biophys. Res. Commun. 148:15-23 (1987)). After these mutations,the specificity for oxaloacetate over pyruvate was increased to 500 incontrast to the wild type enzyme that had a catalytic specificity forpyruvate over oxaloacetate of 1000. This enzyme was further engineeredusing site-directed mutagenesis to have activity towards branched-chainsubstituted pyruvates (Wilks et al., Biochemistry 29:8587-8591 (1990)).Specifically, the enzyme had a 55-fold improvement in K_(cat) foralpha-ketoisocaproate. Three structural modifications were made in thesame enzyme to change its substrate specificity from lactate to malate.The enzyme was highly active and specific towards malate (Wilks et al.,Science 242:1541-1544 (1988)). The same enzyme from B.stearothermophilus was subsequently engineered to have high catalyticactivity towards alpha-keto acids with positively charged side chains,such as those containing ammonium groups (Hogan et al., Biochemistry34:4225-4230 (1995)). Mutants with acidic amino acids introduced atposition 102 of the enzyme favored binding of such side chain ammoniumgroups. The results obtained proved that the mutants showed up to25-fold improvements in k_(cat)/K_(m) values for omega-amino-alpha-ketoacid substrates. Interestingly, this enzyme was also structurallymodified to function as a phenyllactate dehydrogenase instead of alactate dehydrogenase (Wilks et al., Biochemistry 31:7802-7806 1992).Restriction sites were introduced into the gene for the enzyme whichallowed a region of the gene to be excised. This region coded for amobile surface loop of the polypeptide (residues 98-110) which normallyseals the active site from bulk solvent and is a major determinant ofsubstrate specificity. The variable length and sequence loops wereinserted so that hydroxyacid dehydrogenases with altered substratespecificities were generated. With one longer loop construction,activity with pyruvate was reduced one-million-fold but activity withphenylpyruvate was largely unaltered. A switch in specificity(k_(cat)/K_(m)) of 390,000-fold was achieved. The 1700:1 selectivity ofthis enzyme for phenylpyruvate over pyruvate is that required in aphenyllactate dehydrogenase. The studies described above indicate thatvarious approaches of enzyme engineering can be used to obtain enzymesfor the BDO pathways as disclosed herein.

As disclosed herein, biosynthetic pathways to 1,4-butanediol from anumber of central metabolic intermediates are can be utilized, includingacetyl-CoA, succinyl-CoA, alpha-ketoglutarate, glutamate,4-aminobutyrate, and homoserine. Acetyl-CoA, succinyl-CoA andalpha-ketoglutarate are common intermediates of the tricarboxylic acid(TCA) cycle, a series of reactions that is present in its entirety innearly all living cells that utilize oxygen for cellular respiration andis present in truncated forms in a number of anaerobic organisms.Glutamate is an amino acid that is derived from alpha-ketoglutarate viaglutamate dehydrogenase or any of a number of transamination reactions(see FIG. 8B). 4-aminobutyrate can be formed by the decarboxylation ofglutamate (see FIG. 8B) or from acetoacetyl-CoA via the pathwaydisclosed in FIG. 9C. Acetoacetyl-CoA is derived from the condensationof two acetyl-CoA molecules by way of the enzyme, acetyl-coenzyme Aacetyltransferase, or equivalently, acetoacetyl-coenzyme A thiolase.Homoserine is an intermediate in threonine and methionine metabolism,formed from oxaloacetate via aspartate. The conversion of oxaloacetateto homoserine requires one NADH, two NADPH, and one ATP.

Pathways other than those exemplified above also can be employed togenerate the biosynthesis of BDO in non-naturally occurring microbialorganisms. In one embodiment, biosynthesis can be achieved using aL-homoserine to BDO pathway (see FIG. 13). This pathway has a molaryield of 0.90 mol/mol glucose, which appears restricted by theavailability of reducing equivalents. A second pathway synthesizes BDOfrom acetoacetyl-CoA (acetoacetate) and is capable of achieving themaximum theoretical yield of 1.091 mol/mol glucose (see FIG. 9).Implementation of either pathway can be achieved by introduction of twoexogenous enzymes into a host organism, such as E. coli, and bothpathways can additionally complement BDO production via succinyl-CoA.Pathway enzymes, thermodynamics, theoretical yields and overallfeasibility are described further below.

A homoserine pathway also can be engineered to generate BDO-producingmicrobial organisms. Homoserine is an intermediate in threonine andmethionine metabolism, formed from oxaloacetate via aspartate. Theconversion of oxaloacetate to homoserine requires one NADH, two NADPH,and one ATP. Once formed, homoserine feeds into biosynthetic pathwaysfor both threonine and methionine. In most organisms, high levels ofthreonine or methionine feedback to repress the homoserine biosynthesispathway (Caspi et al., Nucleic Acids Res. 34:D511-D516 (1990)).

The transformation of homoserine to 4-hydroxybutyrate (4-HB) can beaccomplished in two enzymatic steps as described herein (see FIG. 13).The first step of this pathway is deamination of homoserine by aputative ammonia lyase. In step 2, the product alkene,4-hydroxybut-2-enoate is reduced to 4-HB by a putative reductase at thecost of one NADH. 4-HB can then be converted to BDO.

Enzymes available for catalyzing the above transformations are disclosedherein. For example, the ammonia lyase in step 1 of the pathway closelyresembles the chemistry of aspartate ammonia-lyase (aspartase).Aspartase is a widespread enzyme in microorganisms, and has beencharacterized extensively (Viola, R. E., Mol. Biol. 74:295-341 (2008)).The crystal structure of the E. coli aspartase has been solved (Shi etal., Biochemistry 36:9136-9144 (1997)), so it is therefore possible todirectly engineer mutations in the enzyme's active site that would alterits substrate specificity to include homoserine. The oxidoreductase instep 2 has chemistry similar to several well-characterized enzymesincluding fumarate reductase in the E. coli TCA cycle. Since thethermodynamics of this reaction are highly favorable, an endogenousreductase with broad substrate specificity will likely be able to reduce4-hydroxybut-2-enoate. The yield of this pathway under anaerobicconditions is 0.9 mol BDO per mol glucose.

The succinyl-CoA pathway was found to have a higher yield due to thefact that it is more energetically efficient. The conversion of oneoxaloacetate molecule to BDO via the homoserine pathway will require theexpenditure of 2 ATP equivalents. Because the conversion of glucose totwo oxaloacetate molecules can generate a maximum of 3 ATP moleculesassuming PEP carboxykinase to be reversible, the overall conversion ofglucose to BDO via homoserine has a negative energetic yield. Asexpected, if it is assumed that energy can be generated via respiration,the maximum yield of the homoserine pathway increases to 1.05 mol/molglucose which is 96% of the succinyl-CoA pathway yield. The succinyl-CoApathway can channel some of the carbon flux through pyruvatedehydrogenase and the oxidative branch of the TCA cycle to generate bothreducing equivalents and succinyl-CoA without an energetic expenditure.Thus, it does not encounter the same energetic difficulties as thehomoserine pathway because not all of the flux is channeled throughoxaloacetate to succinyl-CoA to BDO. Overall, the homoserine pathwaydemonstrates a high-yielding route to BDO.

An acetoacetyl-CoA (acetoacetate) pathway also can be engineered togenerate BDO-producing microbial organisms. Acetoacetyl-CoA(acetoacetate) can be formed from acetyl-CoA by enzymes involved infatty acid metabolism, including acetyl-CoA acetyltransferase andacetoacetyl-CoA transferase. Biosynthetic routes through acetoacetateare also particularly useful in microbial organisms that can metabolizesingle carbon compounds such as carbon monoxide, carbon dioxide ormethanol to form acetyl-CoA.

A three step route from acetoacetyl-CoA (acetoacetate) to4-aminobutyrate (see FIG. 9C) can be used to synthesize BDO throughacetoacetyl-CoA (acetoacetate). 4-Aminobutyrate can be converted tosuccinic semialdehyde as shown in FIG. 8B. Succinic semialdehyde, whichis one reduction step removed from succinyl-CoA or one decarboxylationstep removed from α-ketoglutarate, can be converted to BDO followingthree reductions steps (FIG. 1). Briefly, step 1 of this pathwayinvolves the conversion of acetoacetyl-CoA to acetoacetate by, forexample, the E. coli acetoacetyl-CoA transferase encoded by the atoA andatoD genes (Hanai et al., Appl. Environ. Microbiol. 73:7814-7818(2007)). Step 2 of the acetoacetyl-CoA biopathway entails conversion ofacetoacetate to 3-aminobutanoate by an ω-aminotransferase. The ω-aminoacid:pyruvate aminotransferase (w-APT) from Alcaligens denitrificans wasoverexpressed in E. coli and shown to have a high activity toward3-aminobutanoate in vitro (Yun et al., Appl. Environ. Microbiol.70:2529-2534 (2004)).

In step 3, a putative aminomutase shifts the amine group from the 3- tothe 4-position of the carbon backbone. An aminomutase performing thisfunction on 3-aminobutanoate has not been characterized, but an enzymefrom Clostridium sticklandii has a very similar mechanism. The enzyme,D-lysine-5,6-aminomutase, is involved in lysine biosynthesis.

The synthetic route to BDO from acetoacetyl-CoA (acetoacetate) passesthrough 4-aminobutanoate, a metabolite in E. coli that's normally formedfrom decarboxylation of glutamate. Once formed, 4-aminobutanoate can beconverted to succinic semialdehyde by 4-aminobutanoate transaminase(2.6.1.19), an enzyme which has been biochemically characterized.

One consideration for selecting candidate enzymes in this pathway is thestereoselectivity of the enzymes involved in steps 2 and 3. The ω-ABT inAlcaligens denitrificans is specific to the L-stereoisomer of3-aminobutanoate, while D-lysine-5,6-aminomutase likely requires theD-stereoisomer. If enzymes with complementary stereoselectivity are notinitially found or engineered, a third enzyme can be added to thepathway with racemase activity that can convert L-3-aminobutanoate toD-3-aminobutanoate. While amino acid racemases are widespread, whetherthese enzymes can function on ω-amino acids is not known.

The maximum theoretical molar yield of this pathway under anaerobicconditions is 1.091 mol/mol glucose. In order to generate flux fromacetoacetyl-CoA (acetoacetate) to BDO it was assumed thatacetyl-CoA:acetoacetyl-CoA transferase is reversible. The function ofthis enzyme in E. coli is to metabolize short-chain fatty acids by firstconverting them into thioesters.

While the operation of acetyl-CoA:acetoacetyl-CoA transferase in theacetate-consuming direction has not been demonstrated experimentally inE. coli, studies on similar enzymes in other organisms support theassumption that this reaction is reversible. The enzymebutyryl-CoA:acetate:CoA transferase in gut microbes Roseburia sp. and F.prasnitzii operates in the acetate utilizing direction to producebutyrate (Duncan et al., Appl. Environ. Microbiol 68:5186-5190 (2002)).Another very similar enzyme, acetyl:succinate CoA-transferase inTrypanosoma brucei, also operates in the acetate utilizing direction.This reaction has a Δ_(r×n)G close to equilibrium, so highconcentrations of acetate can likely drive the reaction in the directionof interest. At the maximum theoretical BDO production rate of 1.09mol/mol glucose simulations predict that E. coli can generate 1.098 molATP per mol glucose with no fermentation byproducts. This ATP yieldshould be sufficient for cell growth, maintenance, and production. Theacetoacetyl-CoA (acetoacetate) biopathway is a high-yielding route toBDO from acetyl-CoA.

Therefore, in addition to any of the various modifications exemplifiedpreviously for establishing 4-HB biosynthesis in a selected host, theBDO producing microbial organisms can include any of the previouscombinations and permutations of 4-HB pathway metabolic modifications aswell as any combination of expression for CoA-independent aldehydedehydrogenase, CoA-dependent aldehyde dehydrogenase or an alcoholdehydrogenase or other enzymes disclosed herein to generate biosyntheticpathways for GBL and/or BDO. Therefore, the BDO producers of theinvention can have exogenous expression of, for example, one, two,three, four, five, six, seven, eight, nine, or up to all enzymescorresponding to any of the 4-HB pathway and/or any of the BDO pathwayenzymes disclosed herein.

Design and construction of the genetically modified microbial organismsis carried out using methods well known in the art to achieve sufficientamounts of expression to produce BDO. In particular, the non-naturallyoccurring microbial organisms of the invention can achieve biosynthesisof BDO resulting in intracellular concentrations between about 0.1-200mM or more, such as about 0.1-25 mM or more, as discussed above. Forexample, the intracellular concentration of BDO is between about 3-20mM, particularly between about 5-15 mM and more particularly betweenabout 8-12 mM, including about 10 mM or more. Intracellularconcentrations between and above each of these exemplary ranges also canbe achieved from the non-naturally occurring microbial organisms of theinvention. As with the 4-HB producers, the BDO producers also can besustained, cultured or fermented under anaerobic conditions.

The invention further provides a method for the production of 4-HB. Themethod includes culturing a non-naturally occurring microbial organismhaving a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprisingat least one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate: succinic semialdehyde transaminase,α-ketoglutarate decarboxylase, or glutamate decarboxylase undersubstantially anaerobic conditions for a sufficient period of time toproduce monomeric 4-hydroxybutanoic acid (4-HB). The method canadditionally include chemical conversion of 4-HB to GBL and to BDO orTHF, for example.

Additionally provided is a method for the production of 4-HB. The methodincludes culturing a non-naturally occurring microbial organism having a4-hydroxybutanoic acid (4-HB) biosynthetic pathway including at leastone exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase or α-ketoglutarate decarboxylase under substantiallyanaerobic conditions for a sufficient period of time to producemonomeric 4-hydroxybutanoic acid (4-HB). The 4-HB product can besecreted into the culture medium.

Further provided is a method for the production of BDO. The methodincludes culturing a non-naturally occurring microbial biocatalyst,comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and1,4-butanediol (BDO) biosynthetic pathways, the pathways including atleast one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, succinyl-CoA synthetase, CoA-dependent succinicsemialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase,4-hydroxybutyrate kinase, phosphotranshydroxybutyrylase, α-ketoglutaratedecarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or analdehyde/alcohol dehydrogenase for a sufficient period of time toproduce 1,4-butanediol (BDO). The BDO product can be secreted into theculture medium.

Additionally provided are methods for producing BDO by culturing anon-naturally occurring microbial organism having a BDO pathway of theinvention. The BDO pathway can comprise at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising 4-aminobutyrate CoA transferase,4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoAtransaminase, or 4-hydroxybutyryl-CoA dehydrogenase (see Example VII andTable 17).

Alternatively, the BDO pathway can compare at least one exogenousnucleic acid encoding a BDO pathway enzyme expressed in a sufficientamount to produce BDO, under conditions and for a sufficient period oftime to produce BDO, the BDO pathway comprising 4-aminobutyrate CoAtransferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase,4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoAreductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-oloxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase (seeExample VII and Table 18).

In addition, the invention provides a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising 4-aminobutyrate kinase,4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-oldehydrogenase, 4-aminobutan-1-oloxidoreductase (deaminating),4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acidoxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acidtransaminase, 4-hydroxybutyryl-phosphate dehydrogenase, or4-hydroxybutyraldehyde dehydrogenase (phosphorylating) (see Example VIIand Table 19).

The invention further provides a method for producing BDO, comprisingculturing a non-naturally occurring microbial organism having a BDOpathway, the pathway comprising at least one exogenous nucleic acidencoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising alpha-ketoglutarate 5-kinase,2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating),2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase,alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase,alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic aciddehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming),5-hydroxy-2-oxopentanoic acid decarboxylase, or 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation)(see Example VIII and Table 20).

The invention additionally provides a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising glutamate CoA transferase,glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase,glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoAreductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase(alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase(deaminating), 2-amino-5-hydroxypentanoic acid transaminase,5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoicacid dehydrogenase (decarboxylation)(see Example IX and Table 21).

The invention additionally includes a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising 3-hydroxybutyryl-CoAdehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoAΔ-isomerase, or 4-hydroxybutyryl-CoA dehydratase (see Example X andTable 22).

Also provided is a method for producing BDO, comprising culturing anon-naturally occurring microbial organism having a BDO pathway, thepathway comprising at least one exogenous nucleic acid encoding a BDOpathway enzyme expressed in a sufficient amount to produce BDO, underconditions and for a sufficient period of time to produce BDO, the BDOpathway comprising homoserine deaminase, homoserine CoA transferase,homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoAdeaminase, 4-hydroxybut-2-enoyl-CoA transferase,4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase,4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase,4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or4-hydroxybut-2-enoyl-CoA reductase (see Example XI and Table 23).

The invention additionally provides a method for producing BDO,comprising culturing a non-naturally occurring microbial organism havinga BDO pathway, the pathway comprising at least one exogenous nucleicacid encoding a BDO pathway enzyme expressed in a sufficient amount toproduce BDO, under conditions and for a sufficient period of time toproduce BDO, the BDO pathway comprising succinyl-CoA reductase (alcoholforming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase,4-hydroxybutanal dehydrogenase (phosphorylating)(acylphosphatereductase). Such a BDO pathway can further comprise succinyl-CoAreductase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyryl-CoAtransferase, 4-hydroxybutyrate kinase, phosphotrans-4-hydroxybutyrylase,4-hydroxybutyryl-CoA reductase, 4-hydroxybutyryl-CoA reductase (alcoholforming), or 1,4-butanediol dehydrogenase.

Also provided is a method for producing BDO, comprising culturing anon-naturally occurring microbial organism having a BDO pathway, thepathway comprising at least one exogenous nucleic acid encoding a BDOpathway enzyme expressed in a sufficient amount to produce BDO, underconditions and for a sufficient period of time to produce BDO, the BDOpathway comprising glutamate dehydrogenase, 4-aminobutyrateoxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamatedecarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoAligase, 4-hydroxybutanal dehydrogenase (phosphorylating) (acylphosphatereductase).

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, a 4-HB, BDO, THF or GBL biosynthetic pathway onto the microbialorganism. Alternatively, encoding nucleic acids can be introduced toproduce an intermediate microbial organism having the biosyntheticcapability to catalyze some of the required reactions to confer 4-HB,BDO, THF or GBL biosynthetic capability. For example, a non-naturallyoccurring microbial organism having a 4-HB biosynthetic pathway cancomprise at least two exogenous nucleic acids encoding desired enzymes,such as the combination of 4-hydroxybutanoate dehydrogenase andα-ketoglutarate decarboxylase; 4-hydroxybutanoate dehydrogenase andCoA-independent succinic semialdehyde dehydrogenase; 4-hydroxybutanoatedehydrogenase and CoA-dependent succinic semialdehyde dehydrogenase;CoA-dependent succinic semialdehyde dehydrogenase and succinyl-CoAsynthetase; succinyl-CoA synthetase and glutamate decarboxylase, and thelike. Thus, it is understood that any combination of two or more enzymesof a biosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes of a biosynthetic pathway canbe included in a non-naturally occurring microbial organism of theinvention, for example, 4-hydroxybutanoate dehydrogenase,α-ketoglutarate decarboxylase and CoA-dependent succinic semialdehydedehydrogenase; CoA-independent succinic semialdehyde dehydrogenase andsuccinyl-CoA synthetase; 4-hydroxybutanoate dehydrogenase, CoA-dependentsuccinic semialdehyde dehydrogenase and glutamate:succinic semialdehydetransaminase, and so forth, as desired, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product.

Similarly, for example, with respect to any one or more exogenousnucleic acids introduced to confer BDO production, a non-naturallyoccurring microbial organism having a BDO biosynthetic pathway cancomprise at least two exogenous nucleic acids encoding desired enzymes,such as the combination of 4-hydroxybutanoate dehydrogenase andα-ketoglutarate decarboxylase; 4-hydroxybutanoate dehydrogenase and4-hydroxybutyryl CoA:acetyl-CoA transferase; 4-hydroxybutanoatedehydrogenase and butyrate kinase; 4-hydroxybutanoate dehydrogenase andphosphotransbutyrylase; 4-hydroxybutyryl CoA:acetyl-CoA transferase andaldehyde dehydrogenase; 4-hydroxybutyryl CoA:acetyl-CoA transferase andalcohol dehydrogenase; 4-hydroxybutyryl CoA:acetyl-CoA transferase andan aldehyde/alcohol dehydrogenase, 4-aminobutyrate-CoA transferase and4-aminobutyryl-CoA transaminase; 4-aminobutyrate kinase and4-aminobutan-1-ol oxidoreductase (deaminating), and the like. Thus, itis understood that any combination of two or more enzymes of abiosynthetic pathway can be included in a non-naturally occurringmicrobial organism of the invention. Similarly, it is understood thatany combination of three or more enzymes of a biosynthetic pathway canbe included in a non-naturally occurring microbial organism of theinvention, for example, 4-hydroxybutanoate dehydrogenase,α-ketoglutarate decarboxylase and 4-hydroxybutyryl CoA:acetyl-CoAtransferase; 4-hydroxybutanoate dehydrogenase, butyrate kinase andphosphotransbutyrylase; 4-hydroxybutanoate dehydrogenase,4-hydroxybutyryl CoA:acetyl-CoA transferase and aldehyde dehydrogenase;4-hydroxybutyryl CoA:acetyl-CoA transferase, aldehyde dehydrogenase andalcohol dehydrogenase; butyrate kinase, phosphotransbutyrylase and analdehyde/alcohol dehydrogenase; 4-aminobutyryl-CoA hydrolase,4-aminobutyryl-CoA reductase and 4-amino butan-1-ol transaminase;3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase and4-hydroxybutyryl-CoA dehydratase, and the like. Similarly, anycombination of four, five or more enzymes of a biosynthetic pathway asdisclosed herein can be included in a non-naturally occurring microbialorganism of the invention, as desired, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the 4-HB producers can be cultured for thebiosynthetic production of 4-HB. The 4-HB can be isolated or be treatedas described below to generate GBL, THF and/or BDO. Similarly, the BDOproducers can be cultured for the biosynthetic production of BDO. TheBDO can be isolated or subjected to further treatments for the chemicalsynthesis of BDO family compounds, as disclosed herein.

The growth medium can include, for example, any carbohydrate sourcewhich can supply a source of carbon to the non-naturally occurringmicroorganism. Such sources include, for example, sugars such asglucose, xylose, arabinose, galactose, mannose, fructose, sucrose andstarch. Other sources of carbohydrate include, for example, renewablefeedstocks and biomass. Exemplary types of biomasses that can be used asfeedstocks in the methods of the invention include cellulosic biomass,hemicellulosic biomass and lignin feedstocks or portions of feedstocks.Such biomass feedstocks contain, for example, carbohydrate substratesuseful as carbon sources such as glucose, xylose, arabinose, galactose,mannose, fructose and starch. Given the teachings and guidance providedherein, those skilled in the art will understand that renewablefeedstocks and biomass other than those exemplified above also can beused for culturing the microbial organisms of the invention for theproduction of 4-HB or BDO and other compounds of the invention.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, 4-HB, BDO and any ofthe intermediates metabolites in the 4-HB pathway, the BDO pathwayand/or the combined 4-HB and BDO pathways. All that is required is toengineer in one or more of the enzyme activities shown in FIG. 1 toachieve biosynthesis of the desired compound or intermediate including,for example, inclusion of some or all of the 4-HB and/or BDObiosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that secretes 4-HB when grownon a carbohydrate, secretes BDO when grown on a carbohydrate and/orsecretes any of the intermediate metabolites shown in FIG. 1 when grownon a carbohydrate. The BDO producing microbial organisms of theinvention can initiate synthesis from, for example, succinate,succinyl-CoA, α-ketoglutarate, succinic semialdehyde, 4-HB,4-hydroxybutyrylphosphate, 4-hydroxybutyryl-CoA (4-HB-CoA) and/or4-hydroxybutyraldehyde.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described below in the Examples. Any of these conditions can beemployed with the non-naturally occurring microbial organisms as well asother anaerobic conditions well known in the art. Under such anaerobicconditions, the 4-HB and BDO producers can synthesize monomeric 4-HB andBDO, respectively, at intracellular concentrations of 5-10 mM or more aswell as all other concentrations exemplified previously.

A number of downstream compounds also can be generated for the 4-HB andBDO producing non-naturally occurring microbial organisms of theinvention. With respect to the 4-HB producing microbial organisms of theinvention, monomeric 4-HB and GBL exist in equilibrium in the culturemedium. The conversion of 4-HB to GBL can be efficiently accomplishedby, for example, culturing the microbial organisms in acid pH medium. ApH less than or equal to 7.5, in particular at or below pH 5.5,spontaneously converts 4-HB to GBL.

The resultant GBL can be separated from 4-HB and other components in theculture using a variety of methods well known in the art. Suchseparation methods include, for example, the extraction proceduresexemplified in the Examples as well as methods which include continuousliquid-liquid extraction, pervaporation, membrane filtration, membraneseparation, reverse osmosis, electrodialysis, distillation,crystallization, centrifugation, extractive filtration, ion exchangechromatography, size exclusion chromatography, adsorptionchromatography, and ultrafiltration. All of the above methods are wellknown in the art. Separated GBL can be further purified by, for example,distillation.

Another down stream compound that can be produced from the 4-HBproducing non-naturally occurring microbial organisms of the inventionincludes, for example, BDO. This compound can be synthesized by, forexample, chemical hydrogenation of GBL. Chemical hydrogenation reactionsare well known in the art. One exemplary procedure includes the chemicalreduction of 4-HB and/or GBL or a mixture of these two componentsderiving from the culture using a heterogeneous or homogeneoushydrogenation catalyst together with hydrogen, or a hydride-basedreducing agent used stoichiometrically or catalytically, to produce1,4-butanediol.

Other procedures well known in the art are equally applicable for theabove chemical reaction and include, for example, WO No. 82/03854(Bradley, et al.), which describes the hydrogenolysis ofgamma-butyrolactone in the vapor phase over a copper oxide and zincoxide catalyst. British Pat. No. 1,230,276, which describes thehydrogenation of gamma-butyrolactone using a copper oxide-chromium oxidecatalyst. The hydrogenation is carried out in the liquid phase. Batchreactions also are exemplified having high total reactor pressures.Reactant and product partial pressures in the reactors are well abovethe respective dew points. British Pat. No. 1,314,126, which describesthe hydrogenation of gamma-butyrolactone in the liquid phase over anickel-cobalt-thorium oxide catalyst. Batch reactions are exemplified ashaving high total pressures and component partial pressures well aboverespective component dew points. British Pat. No. 1,344,557, whichdescribes the hydrogenation of gamma-butyrolactone in the liquid phaseover a copper oxide-chromium oxide catalyst. A vapor phase orvapor-containing mixed phase is indicated as suitable in some instances.A continuous flow tubular reactor is exemplified using high totalreactor pressures. British Pat. No. 1,512,751, which describes thehydrogenation of gamma-butyrolactone to 1,4-butanediol in the liquidphase over a copper oxide-chromium oxide catalyst. Batch reactions areexemplified with high total reactor pressures and, where determinable,reactant and product partial pressures well above the respective dewpoints. U.S. Pat. No. 4,301,077, which describes the hydrogenation to1,4-butanediol of gamma-butyrolactone over a Ru—Ni—Co—Zn catalyst. Thereaction can be conducted in the liquid or gas phase or in a mixedliquid-gas phase. Exemplified are continuous flow liquid phase reactionsat high total reactor pressures and relatively low reactorproductivities. U.S. Pat. No. 4,048,196, which describes the productionof 1,4-butanediol by the liquid phase hydrogenation ofgamma-butyrolactone over a copper oxide-zinc oxide catalyst. Furtherexemplified is a continuous flow tubular reactor operating at high totalreactor pressures and high reactant and product partial pressures. AndU.S. Pat. No. 4,652,685, which describes the hydrogenation of lactonesto glycols.

A further downstream compound that can be produced form the 4-HBproducing microbial organisms of the invention includes, for example,THF. This compound can be synthesized by, for example, chemicalhydrogenation of GBL. One exemplary procedure well known in the artapplicable for the conversion of GBL to THF includes, for example,chemical reduction of 4-HB and/or GBL or a mixture of these twocomponents deriving from the culture using a heterogeneous orhomogeneous hydrogenation catalyst together with hydrogen, or ahydride-based reducing agent used stoichiometrically or catalytically,to produce tetrahydrofuran. Other procedures well know in the art areequally applicable for the above chemical reaction and include, forexample, U.S. Pat. No. 6,686,310, which describes high surface areasol-gel route prepared hydrogenation catalysts. Processes for thereduction of maleic acid to tetrahydrofuran (THF) and 1,4-butanediol(BDO) and for the reduction of gamma butyrolactone to tetrahydrofuranand 1,4-butanediol also are described.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described further below in the Examples, particularlyuseful yields of the biosynthetic products of the invention can beobtained under anaerobic or substantially anaerobic culture conditions.

Suitable purification and/or assays to test for the production of 4-HBor BDO can be performed using well known methods. Suitable replicatessuch as triplicate cultures can be grown for each engineered strain tobe tested. For example, product and byproduct formation in theengineered production host can be monitored. The final product andintermediates, and other organic compounds, can be analyzed by methodssuch as HPLC (High Performance Liquid Chromatography), GC-MS (GasChromatography-Mass Spectroscopy) and LC-MS (Liquid Chromatography-MassSpectroscopy) or other suitable analytical methods using routineprocedures well known in the art. The release of product in thefermentation broth can also be tested with the culture supernatant.Byproducts and residual glucose can be quantified by HPLC using, forexample, a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775-779(2005)), or other suitable assay and detection methods well known in theart. The individual enzyme or protein activities from the exogenous DNAsequences can also be assayed using methods well known in the art.

The 4-HB or BDO product can be separated from other components in theculture using a variety of methods well known in the art. Suchseparation methods include, for example, extraction procedures as wellas methods that include continuous liquid-liquid extraction,pervaporation, membrane filtration, membrane separation, reverseosmosis, electrodialysis, distillation, crystallization, centrifugation,extractive filtration, ion exchange chromatography, size exclusionchromatography, adsorption chromatography, and ultrafiltration. All ofthe above methods are well known in the art.

The invention further provides a method of manufacturing 4-HB. Themethod includes fermenting a non-naturally occurring microbial organismhaving a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprisingat least one exogenous nucleic acid encoding 4-hydroxybutanoatedehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,succinyl-CoA synthetase, CoA-dependent succinic semialdehydedehydrogenase, glutamate: succinic semialdehyde transaminase,a-ketoglutarate decarboxylase, or glutamate decarboxylase undersubstantially anaerobic conditions for a sufficient period of time toproduce monomeric 4-hydroxybutanoic acid (4-HB), the process comprisingfed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation.

The culture and chemical hydrogenations described above also can bescaled up and grown continuously for manufacturing of 4-HB, GBL, BDOand/or THF. Exemplary growth procedures include, for example, fed-batchfermentation and batch separation; fed-batch fermentation and continuousseparation, or continuous fermentation and continuous separation. All ofthese processes are well known in the art. Employing the 4-HB producersallows for simultaneous 4-HB biosynthesis and chemical conversion toGBL, BDO and/or THF by employing the above hydrogenation proceduressimultaneous with continuous cultures methods such as fermentation.Other hydrogenation procedures also are well known in the art and can beequally applied to the methods of the invention.

Fermentation procedures are particularly useful for the biosyntheticproduction of commercial quantities of 4-HB and/or BDO. Generally, andas with non-continuous culture procedures, the continuous and/ornear-continuous production of 4-HB or BDO will include culturing anon-naturally occurring 4-HB or BDO producing organism of the inventionin sufficient nutrients and medium to sustain and/or nearly sustaingrowth in an exponential phase. Continuous culture under such conditionscan be include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 ormore weeks and up to several months. Alternatively, organisms of theinvention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of 4-HB, BDO or other 4-HB derivedproducts of the invention can be utilized in, for example, fed-batchfermentation and batch separation; fed-batch fermentation and continuousseparation, or continuous fermentation and continuous separation.Examples of batch and continuous fermentation procedures well known inthe art are exemplified further below in the Examples.

In addition, to the above fermentation procedures using the 4-HB or BDOproducers of the invention for continuous production of substantialquantities of monomeric 4-HB and BDO, respectively, the 4-HB producersalso can be, for example, simultaneously subjected to chemical synthesisprocedures as described previously for the chemical conversion ofmonomeric 4-HB to, for example, GBL, BDO and/or THF. The BDO producerscan similarly be, for example, simultaneously subjected to chemicalsynthesis procedures as described previously for the chemical conversionof BDO to, for example, THF, GBL, pyrrolidones and/or other BDO familycompounds. In addition, the products of the 4-HB and BDO producers canbe separated from the fermentation culture and sequentially subjected tochemical conversion, as disclosed herein.

Briefly, hydrogenation of GBL in the fermentation broth can be performedas described by Frost et al., Biotechnology Progress 18: 201-211 (2002).Another procedure for hydrogenation during fermentation include, forexample, the methods described in, for example, U.S. Pat. No. 5,478,952.This method is further exemplified in the Examples below.

Therefore, the invention additionally provides a method of manufacturingγ-butyrolactone (GBL), tetrahydrofuran (THF) or 1,4-butanediol (BDO).The method includes fermenting a non-naturally occurring microbialorganism having 4-hydroxybutanoic acid (4-HB) and/or 1,4-butanediol(BDO) biosynthetic pathways, the pathways comprise at least oneexogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoAsynthetase, CoA-dependent succinic semialdehyde dehydrogenase,4-hydroxybutyrate:CoA transferase, glutamate: succinic semialdehydetransaminase, α-ketoglutarate decarboxylase, glutamate decarboxylase,4-hydroxybutanoate kinase, phosphotransbutyrylase, CoA-independent1,4-butanediol semialdehyde dehydrogenase, CoA-dependent 1,4-butanediolsemialdehyde dehydrogenase, CoA-independent 1,4-butanediol alcoholdehydrogenase or CoA-dependent 1,4-butanediol alcohol dehydrogenase,under substantially anaerobic conditions for a sufficient period of timeto produce 1,4-butanediol (BDO), GBL or THF, the fermenting comprisingfed-batch fermentation and batch separation; fed-batch fermentation andcontinuous separation, or continuous fermentation and continuousseparation.

In addition to the biosynthesis of 4-HB, BDO and other products of theinvention as described herein, the non-naturally occurring microbialorganisms and methods of the invention also can be utilized in variouscombinations with each other and with other microbial organisms andmethods well known in the art to achieve product biosynthesis by otherroutes. For example, one alternative to produce BDO other than use ofthe 4-HB producers and chemical steps or other than use of the BDOproducer directly is through addition of another microbial organismcapable of converting 4-HB or a 4-HB product exemplified herein to BDO.

One such procedure includes, for example, the fermentation of a 4-HBproducing microbial organism of the invention to produce 4-HB, asdescribed above and below. The 4-HB can then be used as a substrate fora second microbial organism that converts 4-HB to, for example, BDO, GBLand/or THF. The 4-HB can be added directly to another culture of thesecond organism or the original culture of 4-HB producers can bedepleted of these microbial organisms by, for example, cell separation,and then subsequent addition of the second organism to the fermentationbroth can utilized to produce the final product without intermediatepurification steps. One exemplary second organism having the capacity tobiochemically utilize 4-HB as a substrate for conversion to BDO, forexample, is Clostridium acetobutylicum (see, for example, Jewell et al.,Current Microbiology, 13:215-19 (1986)).

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, 4-HB and/or BDO asdescribed. In these embodiments, biosynthetic pathways for a desiredproduct of the invention can be segregated into different microbialorganisms and the different microbial organisms can be co-cultured toproduce the final product. In such a biosynthetic scheme, the product ofone microbial organism is the substrate for a second microbial organismuntil the final product is synthesized. For example, the biosynthesis ofBDO can be accomplished as described previously by constructing amicrobial organism that contains biosynthetic pathways for conversion ofone pathway intermediate to another pathway intermediate or the product,for example, a substrate such as endogenous succinate through 4-HB tothe final product BDO. Alternatively, BDO also can be biosyntheticallyproduced from microbial organisms through co-culture or co-fermentationusing two organisms in the same vessel. A first microbial organism beinga 4-HB producer with genes to produce 4-HB from succinic acid, and asecond microbial organism being a BDO producer with genes to convert4-HB to BDO.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce 4-HB, BDO, GBL and THFproducts of the invention.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of BDO.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene disruption or deletion strategies that result ingenetically stable microorganisms which overproduce the target product.Specifically, the framework examines the complete metabolic and/orbiochemical network of a microorganism in order to suggest geneticmanipulations that force the desired biochemical to become an obligatorybyproduct of cell growth. By coupling biochemical production with cellgrowth through strategically placed gene disruptions or deletions orother functional gene disruptions, for example, deletion of the entiregene, deletion of a regulatory sequence required for transcription ortranslation, deletion of a portion of the gene which results in atruncated gene product, or by any of various mutation strategies thatinactivate the encoded gene product, the growth selection pressuresimposed on the engineered strains after long periods of time in abioreactor lead to improvements in performance as a result of thecompulsory growth-coupled biochemical production. Lastly, when genedeletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or disruptions/deletions.OptKnock computational framework allows the construction of modelformulations that enable an effective query of the performance limits ofmetabolic networks and provides methods for solving the resultingmixed-integer linear programming problems. The metabolic modeling andsimulation methods referred to herein as OptKnock are described in, forexample, U.S. publication 2002/0168654, filed Jan. 10, 2002, inInternational Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S.patent application Ser. No. 11/891,602, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

The methods exemplified above and further illustrated in the Examplesbelow enable the construction of cells and organisms thatbiosynthetically produce, including obligatory couple production of atarget biochemical product to growth of the cell or organism engineeredto harbor the identified genetic alterations. In this regard, metabolicalterations have been identified that result in the biosynthesis of 4-HBand 1,4-butanediol. Microorganism strains constructed with theidentified metabolic alterations produce elevated levels of 4-HB or BDOcompared to unmodified microbial organisms. These strains can bebeneficially used for the commercial production of 4-HB, BDO, THF andGBL, for example, in continuous fermentation process without beingsubjected to the negative selective pressures.

Therefore, the computational methods described herein enable theidentification and implementation of metabolic modifications that areidentified by an in silico method selected from OptKnock or SimPheny®.The set of metabolic modifications can include, for example, addition ofone or more biosynthetic pathway enzymes and/or functional disruption ofone or more metabolic reactions including, for example, disruption bygene deletion.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the BDO producers can be cultured for thebiosynthetic production of BDO.

For the production of BDO, the recombinant strains are cultured in amedium with carbon source and other essential nutrients. It is highlydesirable to maintain anaerobic conditions in the fermenter to reducethe cost of the overall process. Such conditions can be obtained, forexample, by first sparging the medium with nitrogen and then sealing theflasks with a septum and crimp-cap. For strains where growth is notobserved anaerobically, microaerobic conditions can be applied byperforating the septum with a small hole for limited aeration. Exemplaryanaerobic conditions have been described previously and are well-knownin the art. Exemplary aerobic and anaerobic conditions are described,for example, in U.S. patent application Ser. No. 11/891,602, filed Aug.10, 2007. Fermentations can be performed in a batch, fed-batch orcontinuous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

In addition to renewable feedstocks such as those exemplified above, theBDO producing microbial organisms of the invention also can be modifiedfor growth on syngas as its source of carbon. In this specificembodiment, one or more proteins or enzymes are expressed in the BDOproducing organisms to provide a metabolic pathway for utilization ofsyngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:

2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATP

Hence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes or proteins: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes or proteins: methyltetrahydrofolate:corrinoidprotein methyltransferase (for example, AcsE), corrinoid iron-sulfurprotein, nickel-protein assembly protein (for example, AcsF),ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase andnickel-protein assembly protein (for example, CooC). Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate a BDO pathway, thoseskilled in the art will understand that the same engineering design alsocan be performed with respect to introducing at least the nucleic acidsencoding the Wood-Ljungdahl enzymes or proteins absent in the hostorganism. Therefore, introduction of one or more encoding nucleic acidsinto the microbial organisms of the invention such that the modifiedorganism contains the complete Wood-Ljungdahl pathway will confer syngasutilization ability.

Accordingly, given the teachings and guidance provided herein, thoseskilled in the art will understand that a non-naturally occurringmicrobial organism can be produced that secretes the biosynthesizedcompounds of the invention when grown on a carbon source such as acarbohydrate. Such compounds include, for example, BDO and any of theintermediate metabolites in the BDO pathway. All that is required is toengineer in one or more of the required enzyme or protein activities toachieve biosynthesis of the desired compound or intermediate including,for example, inclusion of some or all of the BDO biosynthetic pathways.Accordingly, the invention provides a non-naturally occurring microbialorganism that produces and/or secretes BDO when grown on a carbohydrateor other carbon source and produces and/or secretes any of theintermediate metabolites shown in the BDO pathway when grown on acarbohydrate or other carbon source. The BDO producing microbialorganisms of the invention can initiate synthesis from an intermediatein a BDO pathway, as disclosed herein.

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of BDO.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657(2003)). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Biosynthesis of 4-Hydroxybutanoic Acid

This example describes exemplary biochemical pathways for 4-HBproduction.

Previous reports of 4-HB synthesis in microbes have focused on thiscompound as an intermediate in production of the biodegradable plasticpoly-hydroxyalkanoate (PHA) (U.S. Pat. No. 6,117,658). The use of4-HB/3-HB copolymers over poly-3-hydroxybutyrate polymer (PHB) canresult in plastic that is less brittle (Saito and Doi, Intl. J. Biol.Macromol. 16:99-104 (1994)). The production of monomeric 4-HB describedherein is a fundamentally distinct process for several reasons: (1) theproduct is secreted, as opposed to PHA which is produced intracellularlyand remains in the cell; (2) for organisms that produce hydroxybutanoatepolymers, free 4-HB is not produced, but rather the Coenzyme Aderivative is used by the polyhydroxyalkanoate synthase; (3) in the caseof the polymer, formation of the granular product changesthermodynamics; and (4) extracellular pH is not an issue for productionof the polymer, whereas it will affect whether 4-HB is present in thefree acid or conjugate base state, and also the equilibrium between 4-HBand GBL.

4-HB can be produced in two enzymatic reduction steps from succinate, acentral metabolite of the TCA cycle, with succinic semialdehyde as theintermediate (FIG. 1). The first of these enzymes, succinic semialdehydedehydrogenase, is native to many organisms including E. coli, in whichboth NADH- and NADPH-dependent enzymes have been found (Donnelly andCooper, Eur. J. Biochem. 113:555-561 (1981); Donnelly and Cooper, J.Bacteriol. 145:1425-1427 (1981); Marek and Henson, J. Bacteriol.170:991-994 (1988)). There is also evidence supporting succinicsemialdehyde dehydrogenase activity in S. cerevisiae (Ramos et al., Eur.J. Biochem. 149:401-404 (1985)), and a putative gene has been identifiedby sequence homology. However, most reports indicate that this enzymeproceeds in the direction of succinate synthesis, as shown in FIG. 1(Donnelly and Cooper, supra; Lutke-Eversloh and Steinbuchel, FEMSMicrobiol. Lett. 181:63-71 (1999)), participating in the degradationpathway of 4-HB and gamma-aminobutyrate. Succinic semialdehyde also isnatively produced by certain microbial organisms such as E. coli throughthe TCA cycle intermediate α-ketoglutarate via the action of twoenzymes: glutamate:succinic semialdehyde transaminase and glutamatedecarboxylase. An alternative pathway, used by the obligate anaerobeClostridium kluyveri to degrade succinate, activates succinate tosuccinyl-CoA, then converts succinyl-CoA to succinic semialdehyde usingan alternative succinic semialdehyde dehydrogenase which is known tofunction in this direction (Sohling and Gottschalk, Eur. J. Biochem.212:121-127 (1993)). However, this route has the energetic cost of ATPrequired to convert succinate to succinyl-CoA.

The second enzyme of the pathway, 4-hydroxybutanoate dehydrogenase, isnot native to E. coli or yeast but is found in various bacteria such asC. kluyveri and Ralstonia eutropha (Lutke-Eversloh and Steinbuchel,supra; Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);Valentin et al., Eur. J. Biochem. 227:43-60 (1995); Wolff and Kenealy,Protein Expr. Purif. 6:206-212 (1995)). These enzymes are known to beNADH-dependent, though NADPH-dependent forms also exist. An additionalpathway to 4-HB from alpha-ketoglutarate was demonstrated in E. coliresulting in the accumulation of poly(4-hydroxybutyric acid) (Song etal., Wei Sheng Wu Xue. Bao. 45:382-386 (2005)). The recombinant strainrequired the overexpression of three heterologous genes, PHA synthase(R. eutropha), 4-hydroxybutyrate dehydrogenase (R. eutropha) and4-hydroxybutyrate:CoA transferase (C. kluyveri), along with two nativeE. coli genes: glutamate:succinic semialdehyde transaminase andglutamate decarboxylase. Steps 4 and 5 in FIG. 1 can alternatively becarried out by an alpha-ketoglutarate decarboxylase such as the oneidentified in Euglena gracilis (Shigeoka et al., Biochem. J.282(Pt2):319-323 (1992); Shigeoka and Nakano, Arch. Biochem. Biophys.288:22-28 (1991); Shigeoka and Nakano, Biochem J. 292(Pt 2):463-467(1993)). However, this enzyme has not previously been applied to impactthe production of 4-HB or related polymers in any organism.

The microbial production capabilities of 4-hydroxybutyrate were exploredin two microbes, Escherichia coli and Saccharomyces cerevisiae, using insilico metabolic models of each organism. Potential pathways to 4-HBproceed via a succinate, succinyl-CoA, or alpha-ketoglutarateintermediate as shown in FIG. 1.

A first step in the 4-HB production pathway from succinate involves theconversion of succinate to succinic semialdehyde via an NADH- orNADPH-dependant succinic semialdehyde dehydrogenase. In E. coli, gabD isan NADP-dependant succinic semialdehyde dehydrogenase and is part of agene cluster involved in 4-aminobutyrate uptake and degradation(Niegemann et al., Arch. Microbiol. 160:454-460 (1993); Schneider etal., J. Bacteriol. 184:6976-6986 (2002)). sad is believed to encode theenzyme for NAD-dependant succinic semialdehyde dehydrogenase activity(Marek and Henson, supra). S. cerevisiae contains only theNADPH-dependant succinic semialdehyde dehydrogenase, putatively assignedto UGA2, which localizes to the cytosol (Huh et al., Nature 425:686-691(2003)). The maximum yield calculations assuming the succinate pathwayto 4-HB in both E. coli and S. cerevisiae require only the assumptionthat a non-native 4-HB dehydrogenase has been added to their metabolicnetworks.

The pathway from succinyl-CoA to 4-hydroxybutyrate was described in U.S.Pat. No. 6,117,658 as part of a process for making polyhydroxyalkanoatescomprising 4-hydroxybutyrate monomer units. Clostridium kluyveri is oneexample organism known to possess CoA-dependant succinic semialdehydedehydrogenase activity (Sohling and Gottschalk, supra; Sohling andGottschalk, supra). In this study, it is assumed that this enzyme, fromC. kluyveri or another organism, is expressed in E. coli or S.cerevisiae along with a non-native or heterologous 4-HB dehydrogenase tocomplete the pathway from succinyl-CoA to 4-HB. The pathway fromalpha-ketoglutarate to 4-HB was demonstrated in E. coli resulting in theaccumulation of poly(4-hydroxybutyric acid) to 30% of dry cell weight(Song et al., supra). As E. coli and S. cerevisiae natively orendogenously possess both glutamate:succinic semialdehyde transaminaseand glutamate decarboxylase (Coleman et al., J. Biol. Chem. 276:244-250(2001)), the pathway from AKG to 4-HB can be completed in both organismsby assuming only that a non-native 4-HB dehydrogenase is present.

Example II Biosynthesis of 1,4-Butanediol from Succinate andAlpha-Ketoglutarate

This example illustrates the construction and biosynthetic production of4-HB and BDO from microbial organisms. Pathways for 4-HB and BDO aredisclosed herein.

There are several alternative enzymes that can be utilized in thepathway described above. The native or endogenous enzyme for conversionof succinate to succinyl-CoA (Step 1 in FIG. 1) can be replaced by a CoAtransferase such as that encoded by the cat1 gene C. kluyveri (Sohlingand Gottschalk, Eur. J Biochem. 212:121-127 (1993)), which functions ina similar manner to Step 9. However, the production of acetate by thisenzyme may not be optimal, as it might be secreted rather than beingconverted back to acetyl-CoA. In this respect, it also can be beneficialto eliminate acetate formation in Step 9. As one alternative to this CoAtransferase, a mechanism can be employed in which the 4-HB is firstphosphorylated by ATP and then converted to the CoA derivative, similarto the acetate kinase/phosphotransacetylase pathway in E. coli for theconversion of acetate to acetyl-CoA. The net cost of this route is oneATP, which is the same as is required to regenerate acetyl-CoA fromacetate. The enzymes phosphotransbutyrylase (ptb) and butyrate kinase(bk) are known to carry out these steps on the non-hydroxylatedmolecules for butyrate production in C. acetobutylicum (Cary et al.,Appl Environ Microbiol 56:1576-1583 (1990); Valentine, R. C. and R. S.Wolfe, J Biol Chem. 235:1948-1952 (1960)). These enzymes are reversible,allowing synthesis to proceed in the direction of 4-HB.

BDO also can be produced via α-ketoglutarate in addition to or insteadof through succinate. A described previously, and exemplified furtherbelow, one pathway to accomplish product biosynthesis is with theproduction of succinic semialdehyde via α-ketoglutarate using theendogenous enzymes (FIG. 1, Steps 4-5). An alternative is to use anα-ketoglutarate decarboxylase that can perform this conversion in onestep (FIG. 1, Step 8; Tian et al., Proc Natl Acad Sci US.A102:10670-10675 (2005)).

For the construction of different strains of BDO-producing microbialorganisms, a list of applicable genes was assembled for corroboration.Briefly, one or more genes within the 4-HB and/or BDO biosyntheticpathways were identified for each step of the complete BDO-producingpathway shown in FIG. 1, using available literature resources, the NCBIgenetic database, and homology searches. The genes cloned and assessedin this study are presented below in Table 6, along with the appropriatereferences and URL citations to the polypeptide sequence. As discussedfurther below, some genes were synthesized for codon optimization whileothers were cloned via PCR from the genomic DNA of the native orwild-type organism. For some genes both approaches were used, and inthis case the native genes are indicated by an “n” suffix to the geneidentification number when used in an experiment. Note that only the DNAsequences differ; the proteins are identical.

TABLE 6 Genes expressed in host BDO-producting microbial organisms.Reaction Gene ID number Gene Source number (FIG. 1) name organism Enzymename Link to protein sequence 0001 9 Cat2 Clostridium 4-hydroxybutyratencbi.nlm.nih.gov/entrez/ kluyveri coenzyme A viewer.fcgi?db=nuccore&id=DSM 555 transferase 1228100 0002 12/13 adhE Clostridium Aldehyde/alcoholncbi.nlm.nih.gov/entrez/ acetobutylicum dehydrogenaseviewer.fcgi?db=protein&val= ATCC 824 15004739 0003 12/13 adhE2Clostridium Aldehyde/alcohol ncbi.nlm.nih.gov/entrez/ acetobutylicumdehydrogenase viewer.fcgi?val= ATCC 824 NP_149325.1 0004 1 Cat1Clostridium Succinate ncbi.nlm.nih.gov/entrez/ kluyveri coenzyme Aviewer.fcgi?db=nuccore&id= DSM 555 transferase 1228100 0008 6 sucDClostridium Succinic ncbi.nlm.nih.gov/entrez/ kluyveri semialdehydeviewer.fcgi?db=nuccore&id= DSM 555 dehydrogenase 1228100 (CoA-dependent)0009 7 4-HBd Ralstonia 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/eutropha H16 dehydrogenase viewer.fcgi?val= (NAD-dependent) YP_726053.10010 7 4-HBd Clostridium 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/kluyveri dehydrogenase viewer.fcgi?db=nuccore&id= DSM 555(NAD-dependent) 1228100 0011 12/13 adhE E. coli Aldehyde/alcoholshigen.nig.ac.jp/ecoli/pec/ dehydrogenase genes.List.DetailAction.do?fromListFlag=true&feature Type=1&orfId=1219 0012 12/13 yqhD E. coliAldehyde alcohol shigen.nig.ac.jp/ecoli/pec/ dehydrogenasegenes.List.DetailAction.do 0013 13 bdhB Clostridium Butanolncbi.nlm.nih.gov/entrez/ acetobutylicum dehydrogenase IIviewer.fcgi?val=NP_349891.1 ATCC 824 0020 11 ptb Clostridium Phospho-ncbi.nlm.nih.gov/entrez/ acetobutylicum transbutyrylaseviewer.fcgi?db=protein&id= ATCC 824 15896327 0021 10 buk1 ClostridiumButyrate kinase I ncbi.nlm.nih.gov/entrez/ acetobutylicumviewer.fcgi?db=protein&id= ATCC 824 20137334 0022 10 buk2 ClostridiumButyrate kinase II ncbi.nlm.nih.gov/entrez/ acetobutylicumviewer.fcgi?db=protein&id= ATCC 824 20137415 0023 13 adhEm isolated fromAlcohol metalibrary dehydrogenase of anaerobic sewage digester microbialconsortia 0024 13 adhE Clostridium Alcohol genome.jp/dbget- thermocellumdehydrogenase bin/wwwbget?cth:Cthe_0423 0025 13 ald Clostridium CoenzymeA- ncbi.nlm.nih.gov/entrez/ beijerinckii acylating aldehydeviewer.fcgi?db=protein&id= dehydrogenase 49036681 0026 13 bdhAClostridium Butanol ncbi.nlm.nih.gov/entrez/ acetobutylicumdehydrogenase viewer.fcgi?val=NP_349892.1 ATCC 824 0027 12 bldClostridium Butyraldehyde ncbi.nlm.nih.gov/entrez/ saccharoper-dehydrogenase viewer.fcgi?db=protein&id= butylacetonicum 31075383 002813 bdh Clostridium Butanol ncbi.nlm.nih.gov/entrez/ saccharoper-dehydrogenase viewer.fcgi?db=protein&id= butylacetonicum 124221917 002912/13 adhE Clostridium Aldehyde/alcohol genome.jp/dbget- tetanidehydrogenase bin/www_bget?ctc:CTC01366 0030 12/13 adhE ClostridiumAldehyde/alcohol genome.jp/dbget- perfringens dehydrogenasebin/www_bget?cpe:CPE2531 0031 12/13 adhE Clostridium Aldehyde/alcoholgenome.jp/dbget- difficile dehydrogenase bin/www_bget?cdf:CD2966 0032 8sucA Mycobacterium α-ketoglutarate ncbi.nlm.nih.gov/entrez/ bovisdecarboxylase viewer.fcgi?val=YP_977400.1 BCG, Pasteur 0033 9 cat2Clostridium 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/ aminobutyricumcoenzyme A viewer.fcgi?db=protein&val= transferase 6249316 0034 9 cat2Porphyromonas 4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/ gingivaliscoenzyme A viewer.fcgi?db=protein&val= W83 transferase 34541558 0035 6sucD Porphyromonas Succinic ncbi.nlm.nih.gov/entrez/ gingivalissemialdehyde viewer.fcgi?val=NP_904963.1 W83 dehydrogenase(CoA-dependent) 0036 7 4-HBd Porphyromonas NAD-dependentncbi.nlm.nih.gov/entrez/ gingivalis 4-hydroxybutyrateviewer.fcgi?val=NP_904964.1 W83 dehydrogenase 0037 7 gbd Uncultured4-hydroxybutyrate ncbi.nlm.nih.gov/entrez/ bacterium dehydrogenaseviewer.fcgi?db=nuccore&id= 5916168 0038 1 sucCD E. coli Succinyl-CoAshigen.nig.ac.jp/ecoli/pec/ synthetase genes.List.DetailAction.do

Expression Vector Construction for BDO Pathway.

Vector backbones and some strains were obtained from Dr. Rolf Lutz ofExpressys (expressys.de). The vectors and strains are based on the pZExpression System developed by Dr. Rolf Lutz and Prof Hermann Bujard(Lutz, R. and H. Bujard, Nucleic Acids Res 25:1203-1210 (1997)). Vectorsobtained were pZE13luc, pZA33luc, pZS* 13luc and pZE22luc and containedthe luciferase gene as a stuffer fragment. To replace the luciferasestuffer fragment with a lacZ-alpha fragment flanked by appropriaterestriction enzyme sites, the luciferase stuffer fragment was firstremoved from each vector by digestion with EcoRI and XbaI. ThelacZ-alpha fragment was PCR amplified from pUC19 with the followingprimers:

lacZalpha-RI (SEQ ID NO: 1)5′GACGAATTCGCTAGCAAGAGGAGAAGTCGACATGTCCAATTCACTGG CCGTCGTTTTAC3′lacZalpha 3′BB (SEQ ID NO: 2)5′-GACCCTAGGAAGCTTTCTAGAGTCGACCTATGCGGCATCAGAGCAG A-3′.

This generated a fragment with a 5′ end of EcoRI site, NheI site, aRibosomal Binding Site, a SalI site and the start codon. On the 3′ endof the fragment contained the stop codon, XbaI, HindIII, and AvrIIsites. The PCR product was digested with EcoRI and AvrII and ligatedinto the base vectors digested with EcoRI and XbaI (XbaI and AvrII havecompatible ends and generate a non-site). Because NheI and XbaIrestriction enzyme sites generate compatible ends that can be ligatedtogether (but generate a NheI/XbaI non-site that is not digested byeither enzyme), the genes cloned into the vectors could be “Biobricked”together (openwetware.org/wiki/Synthetic_Biology:BioBricks). Briefly,this method enables joining an unlimited number of genes into the vectorusing the same 2 restriction sites (as long as the sites do not appearinternal to the genes), because the sites between the genes aredestroyed after each addition.

All vectors have the pZ designation followed by letters and numbersindication the origin of replication, antibiotic resistance marker andpromoter/regulatory unit. The origin of replication is the second letterand is denoted by E for ColE1, A for p15A and S for pSC101-basedorigins. The first number represents the antibiotic resistance marker (1for Ampicillin, 2 for Kanamycin, 3 for Chloramphenicol, 4 forSpectinomycin and 5 for Tetracycline). The final number defines thepromoter that regulated the gene of interest (1 for P_(LtetO-1), 2 forP_(LlacO-1), 3 for P_(A1lacO-1), and 4 for P_(lac/ara-1)). The MCS andthe gene of interest follows immediately after. For the work discussedhere we employed two base vectors, pZA33 and pZE13, modified for thebiobricks insertions as discussed above. Once the gene(s) of interesthave been cloned into them, resulting plasmids are indicated using thefour digit gene codes given in Table 6; e.g., pZA33-XXXX-YYYY- . . . .

Host Strain Construction.

The parent strain in all studies described here is E. coli K-12 strainMG1655. Markerless deletion strains in adhE, gabD, and aldA wereconstructed under service contract by a third party using the redETmethod (Datsenko, K. A. and B. L. Wanner, Proc Natl Acad Sci US.A97:6640-6645 (2000)). Subsequent strains were constructed viabacteriophage P1 mediated transduction (Miller, J. Experiments inMolecular Genetics, Cold Spring Harbor Laboratories, New York (1973)).Strain C600Z1 (laci^(q), PN25-tetR, Sp^(R), lacY1, leuB6, mcrB+, supE44,thi-1, thr-1, tonA21) was obtained from Expressys and was used as asource of a lacI^(q) allele for P1 transduction. Bacteriophage P1vir wasgrown on the C600Z1 E. coli strain, which has the spectinomycinresistance gene linked to the lacI^(q). The P1 lysate grown on C600Z1was used to infect MG1655 with selection for spectinomycin resistance.The spectinomycin resistant colonies were then screened for the linkedlacI^(q) by determining the ability of the transductants to repressexpression of a gene linked to a P_(A1lacO-1) promoter. The resultingstrain was designated MG1655 lacI^(q). A similar procedure was used tointroduce lacI^(Q) into the deletion strains.

Production of 4-HB from Succinate.

For construction of a 4-HB producer from succinate, genes encoding stepsfrom succinate to 4-HB and 4-HB-CoA (1, 6, 7, and 9 in FIG. 1) wereassembled onto the pZA33 and pZE13 vectors as described below. Variouscombinations of genes were assessed, as well as constructs bearingincomplete pathways as controls (Tables 7 and 8). The plasmids were thentransformed into host strains containing lacI^(Q), which allow inducibleexpression by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).Both wild-type and hosts with deletions in genes encoding the nativesuccinic semialdehyde dehydrogenase (step 2 in FIG. 1) were tested.

Activity of the heterologous enzymes were first tested in in vitroassays, using strain MG1655 lacI^(Q) as the host for the plasmidconstructs containing the pathway genes. Cells were grown aerobically inLB media (Difco) containing the appropriate antibiotics for eachconstruct, and induced by addition of IPTG at 1 mM when the opticaldensity (OD600) reached approximately 0.5. Cells were harvested after 6hours, and enzyme assays conducted as discussed below.

In Vitro Enzyme Assays.

To obtain crude extracts for activity assays, cells were harvested bycentrifugation at 4,500 rpm (Beckman-Coulter, Allegera X-15R) for 10min. The pellets were resuspended in 0.3 mL BugBuster (Novagen) reagentwith benzonase and lysozyme, and lysis proceeded for 15 minutes at roomtemperature with gentle shaking. Cell-free lysate was obtained bycentrifugation at 14,000 rpm (Eppendorf centrifuge 5402) for 30 min at4° C. Cell protein in the sample was determined using the method ofBradford et al., Anal. Biochem. 72:248-254 (1976), and specific enzymeassays conducted as described below. Activities are reported in Units/mgprotein, where a unit of activity is defined as the amount of enzymerequired to convert 1 μmol of substrate in 1 min. at room temperature.In general, reported values are averages of at least 3 replicate assays.

Succinyl-CoA transferase (Cat1) activity was determined by monitoringthe formation of acetyl-CoA from succinyl-CoA and acetate, following apreviously described procedure Sohling and Gottschalk, J. Bacteriol.178:871-880 (1996). Succinyl-CoA synthetase (SucCD) activity wasdetermined by following the formation of succinyl-CoA from succinate andCoA in the presence of ATP. The experiment followed a proceduredescribed by Cha and Parks, J. Biol. Chem. 239:1961-1967 (1964).CoA-dependent succinate semialdehyde dehydrogenase (SucD) activity wasdetermined by following the conversion of NAD to NADH at 340 nm in thepresence of succinate semialdehyde and CoA (Sohling and Gottschalk, Eur.J. Biochem. 212:121-127 (1993)). 4-HB dehydrogenase (4-HBd) enzymeactivity was determined by monitoring the oxidation of NADH to NAD at340 nm in the presence of succinate semialdehyde. The experimentfollowed a published procedure Gerhardt et al. Arch. Microbiol.174:189-199 (2000). 4-HB CoA transferase (Cat2) activity was determinedusing a modified procedure from Scherf and Buckel, Appl. Environ.Microbiol. 57:2699-2702 (1991). The formation of 4-HB-CoA or butyryl-CoAformation from acetyl-CoA and 4-HB or butyrate was determined usingHPLC.

Alcohol (ADH) and aldehyde (ALD) dehydrogenase was assayed in thereductive direction using a procedure adapted from several literaturesources (Durre et al., FEMS Microbiol. Rev. 17:251-262 (1995); Palosaariand Rogers, J. Bacteriol. 170:2971-2976 (1988) and Welch et al., Arch.Biochem. Biophys. 273:309-318 (1989). The oxidation of NADH is followedby reading absorbance at 340 nM every four seconds for a total of 240seconds at room temperature. The reductive assays were performed in 100mM MOPS (adjusted to pH 7.5 with KOH), 0.4 mM NADH, and from 1 to 50 μlof cell extract. The reaction is started by adding the followingreagents: 100 μl of 100 mM acetaldehyde or butyraldehyde for ADH, or 100μl of 1 mM acetyl-CoA or butyryl-CoA for ALD. The Spectrophotometer isquickly blanked and then the kinetic read is started. The resultingslope of the reduction in absorbance at 340 nM per minute, along withthe molar extinction coefficient of NAD(P)H at 340 nM (6000) and theprotein concentration of the extract, can be used to determine thespecific activity.

The enzyme activity of PTB is measured in the direction of butyryl-CoAto butyryl-phosphate as described in Cary et al. J. Bacteriol.170:4613-4618 (1988). It provides inorganic phosphate for theconversion, and follows the increase in free CoA with the reagent5,5′-dithiobis-(2-nitrobenzoic acid), or DTNB. DTNB rapidly reacts withthiol groups such as free CoA to release the yellow-colored2-nitro-5-mercaptobenzoic acid (TNB), which absorbs at 412 nm with amolar extinction coefficient of 14,140 M cm⁻¹. The assay buffercontained 150 mM potassium phosphate at pH 7.4, 0.1 mM DTNB, and 0.2 mMbutyryl-CoA, and the reaction was started by addition of 2 to 50 μL cellextract. The enzyme activity of BK is measured in the direction ofbutyrate to butyryl-phosphate formation at the expense of ATP. Theprocedure is similar to the assay for acetate kinase previouslydescribed Rose et al., J. Biol. Chem. 211:737-756 (1954). However wehave found another acetate kinase enzyme assay protocol provided bySigma to be more useful and sensitive. This assay links conversion ofATP to ADP by acetate kinase to the linked conversion of ADP andphosphoenol pyruvate (PEP) to ATP and pyruvate by pyruvate kinase,followed by the conversion of pyruvate and NADH to lactate and NAD+ bylactate dehydrogenase. Substituting butyrate for acetate is the onlymajor modification to enable the assay to follow BK enzyme activity. Theassay mixture contained 80 mM triethanolamine buffer at pH 7.6, 200 mMsodium butyrate, 10 mM MgCl2, 0.1 mM NADH, 6.6 mM ATP, 1.8 mMphosphoenolpyruvate. Pyruvate kinase, lactate dehydrogenase, andmyokinase were added according to the manufacturer's instructions. Thereaction was started by adding 2 to 50 μL cell extract, and the reactionwas monitored based on the decrease in absorbance at 340 nm indicatingNADH oxidation.

Analysis of CoA Derivatives by HPLC.

An HPLC based assay was developed to monitor enzymatic reactionsinvolving coenzyme A (CoA) transfer. The developed method enabled enzymeactivity characterization by quantitative determination of CoA, acetylCoA (AcCoA), butyryl CoA (BuCoA) and 4-hydroxybutyrate CoA (4-HBCoA)present in in-vitro reaction mixtures. Sensitivity down to low μM wasachieved, as well as excellent resolution of all the CoA derivatives ofinterest.

Chemical and sample preparation was performed as follows. Briefly, CoA,AcCoA, BuCoA and all other chemicals, were obtained from Sigma-Aldrich.The solvents, methanol and acetonitrile, were of HPLC grade. Standardcalibration curves exhibited excellent linearity in the 0.01-1 mg/mLconcentration range. Enzymatic reaction mixtures contained 100 mM TrisHCl buffer (pH 7), aliquots were taken at different time points,quenched with formic acid (0.04% final concentration) and directlyanalyzed by HPLC.

HPLC analysis was performed using an Agilent 1100 HPLC system equippedwith a binary pump, degasser, thermostated autosampler and columncompartment, and diode array detector (DAD), was used for the analysis.A reversed phase column, Kromasil 100 5 um C18, 4.6×150 mm (PeekeScientific), was employed. 25 mM potassium phosphate (pH 7) and methanolor acetonitrile, were used as aqueous and organic solvents at 1 mL/minflow rate. Two methods were developed: a short one with a fastergradient for the analysis of well-resolved CoA, AcCoA and BuCoA, and alonger method for distinguishing between closely eluting AcCoA and4-HBCoA. Short method employed acetonitrile gradient (0 min—5%, 6min—30%, 6.5 min—5%, 10 min—5%) and resulted in the retention times 2.7,4.1 and 5.5 min for CoA, AcCoA and BuCoA, respectively. In the longmethod methanol was used with the following linear gradient: 0 min—5%,20 min—35%, 20.5 min—5%, 25 min—5%. The retention times for CoA, AcCoA,4-HBCoA and BuCoA were 5.8, 8.4, 9.2 and 16.0 min, respectively. Theinjection volume was 5 μL, column temperature 30° C., and UV absorbancewas monitored at 260 nm.

The results demonstrated activity of each of the four pathway steps(Table 7), though activity is clearly dependent on the gene source,position of the gene in the vector, and the context of other genes withwhich it is expressed. For example, gene 0035 encodes a succinicsemialdehyde dehydrogenase that is more active than that encoded by0008, and 0036 and 0010n are more active 4-HB dehydrogenase genes than0009. There also seems to be better 4-HB dehydrogenase activity whenthere is another gene preceding it on the same operon.

TABLE 7 In vitro enzyme activities in cell extracts from MG1655 lacI^(Q)containing the plasmids expressing genes in the 4-HB-CoA pathway.Activities are reported in Units/mg protein, where a unit of activity isdefined as the amount of enzyme required to convert l μmol of substratein 1 min. at room temperature. Sample Cell # pZE13 (a) pZA33 (b) OD600Prot (c) Cat1 SucD 4HBd Cat2 1 cat1 (0004) 2.71 6.43 1.232 0.00 2 cat1(0004)-sucD (0035) 2.03 5.00 0.761 2.57 3 cat1 (0004)-sucD (0008) 1.043.01 0.783 0.01 4 sucD (0035) 2.31 6.94 2.32 5 sucD (0008) 1.10 4.160.05 6 4hbd (0009) 2.81 7.94 0.003 0.25 7 4hbd (0036) 2.63 7.84 3.31 84hbd (0010n) 2.00 5.08 2.57 9 cat1 (0004)-sucD (0035) 4hbd (0009) 2.075.04 0.600 1.85 0.01 10 cat1 (0004)-sucD (0035) 4hbd (0036) 2.08 5.400.694 1.73 0.41 11 cat1 (0004)-sucD (0035) 4hbd (0010n) 2.44 4.73 0.6792.28 0.37 12 cat1 (0004)-sucD (0008) 4hbd (0009) 1.08 3.99 0.572 −0.010.02 13 cat1 (0004)-sucD (0008) 4hbd (0036) 0.77 2.60 0.898 −0.01 0.0414 cat1 (0004)-sucD (0008) 4hbd (0010n) 0.63 2.47 0.776 0.00 0.00 15cat2 (0034) 2.56 7.86 1.283 16 cat2(0034)-4hbd(0036) 3.13 8.04 24.860.993 17 cat2(0034)-4hbd(0010n) 2.38 7.03 7.45 0.675 184hbd(0036)-cat2(0034) 2.69 8.26 2.15 7.490 19 4hbd(0010n)-cat2(0034)2.44 6.59 0.59 4.101 (a) Genes expressed from Plac on pZE13, a high-copyplasmid with colE1 origin and ampicillin resistance. Gene identificationnumbers are as given in Table 6 (b) Genes expressed from Plac on pZA33,a medium-copy plasmid with pACYC origin and chloramphenicol resistance.(c) Cell protein given as mg protein per mL extract.

Recombinant strains containing genes in the 4-HB pathway were thenevaluated for the ability to produce 4-HB in vivo from central metabolicintermediates. Cells were grown anaerobically in LB medium to OD600 ofapproximately 0.4, then induced with 1 mM IPTG. One hour later, sodiumsuccinate was added to 10 mM, and samples taken for analysis followingan additional 24 and 48 hours. 4-HB in the culture broth was analyzed byGC-MS as described below. The results indicate that the recombinantstrain can produce over 2 mM 4-HB after 24 hours, compared toessentially zero in the control strain (Table 8).

TABLE 8 Production of 4-HB from succinate in E. coli strains harboringplasmids expressing various combinations of 4-HB pathway genes. 24 Hours48 Hours Sample 4HB, 4HB 4HB, 4HB # Host Strain pZE13 pZA33 OD600 μMnorm. (a) OD600 μM norm. (a) 1 MG1655 lacIq cat1 (0004)-sucD (0035) 4hbd(0009) 0.47 487 1036 1.04 1780 1711 2 MG1655 lacIq cat1 (0004)-sucD(0035) 4hbd (0027) 0.41 111 270 0.99 214 217 3 MG1655 lacIq cat1(0004)-sucD (0035) 4hbd (0036) 0.47 863 1835 0.48 2152 4484 4 MG1655lacIq cat1 (0004)-sucD (0035) 4hbd (0010n) 0.46 956 2078 0.49 2221 45335 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0009) 0.38 493 1296 0.371338 3616 6 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0027) 0.32 26 810.27 87 323 7 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0036) 0.24 5062108 0.31 1448 4672 8 MG1655 lacIq cat1 (0004)-sucD (0008) 4hbd (0010n)0.24 78 324 0.56 233 416 9 MG1655 lacIq gabD cat1 (0004)-sucD (0035)4hbd (0009) 0.53 656 1237 1.03 1643 1595 10 MG1655 lacIq gabD cat1(0004)-sucD (0035) 4hbd (0027) 0.44 92 209 0.98 214 218 11 MG1655 lacIqgabD cat1 (0004)-sucD (0035) 4hbd (0036) 0.51 1072 2102 0.97 2358 243112 MG1655 lacIq gabD cat1 (0004)-sucD (0035) 4hbd (0010n) 0.51 981 19240.97 2121 2186 13 MG1655 lacIq gabD cat1 (0004)-sucD (0008) 4hbd (0009)0.35 407 1162 0.77 1178 1530 14 MG1655 lacIq gabD cat1 (0004)-sucD(0008) 4hbd (0027) 0.51 19 36 1.07 50 47 15 MG1655 lacIq gabD cat1(0004)-sucD (0008) 4hbd (0036) 0.35 584 1669 0.78 1350 1731 16 MG1655lacIq gabD cat1 (0004)-sucD (0008) 4hbd (0010n) 0.32 74 232 0.82 232 28317 MG1655 lacIq vector only vector only 0.8 1 2 1.44 3 2 18 MG1655 lacIqgabD vector only vector only 0.89 1 2 1.41 7 5 (a) Normalized 4-HBconcentration, μM/OD600 units

An alternate to using a CoA transferase (cat1) to produce succinyl-CoAfrom succinate is to use the native E. coli sucCD genes, encodingsuccinyl-CoA synthetase. This gene cluster was cloned onto pZE13 alongwith candidate genes for the remaining steps to 4-HB to createpZE13-0038-0035-0036.

Production of 4-HB from Glucose.

Although the above experiments demonstrate a functional pathway to 4-HBfrom a central metabolic intermediate (succinate), an industrial processwould require the production of chemicals from low-cost carbohydratefeedstocks such as glucose or sucrose. Thus, the next set of experimentswas aimed to determine whether endogenous succinate produced by thecells during growth on glucose could fuel the 4-HB pathway. Cells weregrown anaerobically in M9 minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/LKH₂PO₄, 0.5 g/L NaCl, 1.0 g/L NH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂)supplemented with 20 g/L glucose, 100 mM 3-(N-morpholino)propanesulfonicacid (MOPS) to improve the buffering capacity, 10 μg/mL thiamine, andthe appropriate antibiotics. 0.25 mM IPTG was added when OD600 reachedapproximately 0.2, and samples taken for 4-HB analysis every 24 hoursfollowing induction. In all cases 4-HB plateaued after 24 hours, with amaximum of about 1 mM in the best strains (FIG. 3 a), while thesuccinate concentration continued to rise (FIG. 3 b). This indicatesthat the supply of succinate to the pathway is likely not limiting, andthat the bottleneck may be in the activity of the enzymes themselves orin NADH availability. 0035 and 0036 are clearly the best gene candidatesfor CoA-dependent succinic semialdehyde dehydrogenase and 4-HBdehydrogenase, respectively. The elimination of one or both of the genesencoding known (gabD) or putative (aldA) native succinic semialdehydedehydrogenases had little effect on performance. Finally, it should benoted that the cells grew to a much lower OD in the 4-HB-producingstrains than in the controls (FIG. 3 c).

An alternate pathway for the production of 4-HB from glucose is viaa-ketoglutarate. We explored the use of an α-ketoglutarate decarboxylasefrom Mycobacterium tuberculosis Tian et al., Proc. Natl. Acad. Sci. USA102:10670-10675 (2005) to produce succinic semialdehyde directly fromα-ketoglutarate (step 8 in FIG. 1). To demonstrate that this gene (0032)was functional in vivo, we expressed it on pZE13 in the same host as4-HB dehydrogenase (gene 0036) on pZA33. This strain was capable ofproducing over 1.0 mM 4-HB within 24 hours following induction with 1 mMIPTG (FIG. 4). Since this strain does not express a CoA-dependentsuccinic semialdehyde dehydrogenase, the possibility of succinicsemialdehyde production via succinyl-CoA is eliminated. It is alsopossible that the native genes responsible for producing succinicsemialdehyde could function in this pathway (steps 4 and 5 in FIG. 1);however, the amount of 4-HB produced when the pZE13-0032 plasmid wasleft out of the host is the negligible.

Production of BDO from 4-HB.

The production of BDO from 4-HB required two reduction steps, catalyzedby dehydrogenases. Alcohol and aldehyde dehydrogenases (ADH and ALD,respectively) are NAD+/H and/or NADP+/H-dependent enzymes that togethercan reduce a carboxylic acid group on a molecule to an alcohol group, orin reverse, can perform the oxidation of an alcohol to a carboxylicacid. This biotransformation has been demonstrated in wild-typeClostridium acetobutylicum (Jewell et al., Current Microbiology,13:215-19 (1986)), but neither the enzymes responsible nor the genesresponsible were identified. In addition, it is not known whetheractivation to 4-HB-CoA is first required (step 9 in FIG. 1), or if thealdehyde dehydrogenase (step 12) can act directly on 4-HB. We developeda list of candidate enzymes from C. acetobutylicum and related organismsbased on known activity with the non-hydroxylated analogues to 4-HB andpathway intermediates, or by similarity to these characterized genes(Table 6). Since some of the candidates are multifunctionaldehydrogenases, they could potentially catalyze both theNAD(P)H-dependent reduction of the acid (or CoA-derivative) to thealdehyde, and of the aldehyde to the alcohol. Before beginning work withthese genes in E. coli, we first validated the result referenced aboveusing C. acetobutylicum ATCC 824. Cells were grown in Schaedler broth(Accumedia, Lansing, Mich.) supplemented with 10 mM 4-HB, in ananaerobic atmosphere of 10% CO₂, 10% H₂, and 80% N₂ at 30° C. Periodicculture samples were taken, centrifuged, and the broth analyzed for BDOby GC-MS as described below. BDO concentrations of 0.1 mM, 0.9 mM, and1.5 mM were detected after 1 day, 2 days, and 7 days incubation,respectively. No BDO was detected in culture grown without 4-HBaddition. To demonstrate that the BDO produced was derived from glucose,we grew the best BDO producing strain MG1655 lacI^(Q)pZE13-0004-0035-0002 pZA33-0034-0036 in M9 minimal medium supplementedwith 4 g/L uniformly labeled ¹³C-glucose. Cells were induced at OD of0.67 with 1 mM IPTG, and a sample taken after 24 hours. Analysis of theculture supernatant was performed by mass spectrometry.

Gene candidates for the 4-HB to BDO conversion pathway were next testedfor activity when expressed in the E. coli host MG1655 lacI^(Q).Recombinant strains containing each gene candidate expressed on pZA33were grown in the presence of 0.25 mM IPTG for four hours at 37° C. tofully induce expression of the enzyme. Four hours after induction, cellswere harvested and assayed for ADH and ALD activity as described above.Since 4-HB-CoA and 4-hydroxybutyraldehyde are not availablecommercially, assays were performed using the non-hydroxylatedsubstrates (Table 9). The ratio in activity between 4-carbon and2-carbon substrates for C. acetobutylicum adhE2 (0002) and E. coli adhE(0011) were similar to those previously reported in the literature aAtsumi et al., Biochim. Biophys. Acta. 1207:1-11 (1994).

TABLE 9 In vitro enzyme activities in cell extracts from MG1655 lacI^(Q)containing pZA33 expressing gene candidates for aldehyde and alcoholdehydrogenases. Activities are expressed in μmol min⁻¹ mg cellprotein⁻¹. Aldehyde dehydrogenase Alcohol dehydrogenase Butyryl- Acetyl-Butyral- Acetal- Gene Substrate CoA CoA dehyde dehyde 0002 0.0076 0.00460.0264 0.0247 0003n 0.0060 0.0072 0.0080 0.0075 0011 0.0069 0.00950.0265 0.0093 0013 N.D. N.D. 0.0130 0.0142 0023 0.0089 0.0137 0.01780.0235 0025 0 0.0001 N.D. N.D. 0026 0 0.0005 0.0024 0.0008 N.D., notdetermined.

For the BDO production experiments, cat2 from Porphyromonas gingivalisW83 (gene 0034) was included on pZA33 for the conversion of 4-HB to4-HB-CoA, while the candidate dehydrogenase genes were expressed onpZE13. The host strain was MG1655 lacI^(Q). Along with the alcohol andaldehyde dehydrogenase candidates, we also tested the ability ofCoA-dependent succinic semialdehyde dehydrogenases (sucD) to function inthis step, due to the similarity of the substrates. Cells were grown toan OD of about 0.5 in LB medium supplemented with 10 mM 4-HB, inducedwith 1 mM IPTG, and culture broth samples taken after 24 hours andanalyzed for BDO as described below. The best BDO production occurredusing adhE2 from C. acetobutylicum, sucD from C. kluyveri, or sucD fromP. gingivalis (FIG. 5). Interestingly, the absolute amount of BDOproduced was higher under aerobic conditions; however, this is primarilydue to the lower cell density achieved in anaerobic cultures. Whennormalized to cell OD, the BDO production per unit biomass is higher inanaerobic conditions (Table 10).

TABLE 10 Absolute and normalized BDO concentrations from cultures ofcells expressing adhE2 from C. acetobutylicum, sucD from C. kluyveri, orsucD from P. gingivalis (data from experiments 2, 9, and 10 in FIG. 3),as well as the negative control (experiment 1). Gene BDO OD expressedConditions (μM) (600 nm) BDO/OD none Aerobic 0 13.4 0 none Microaerobic0.5 6.7 0.09 none Anaerobic 2.2 1.26 1.75 0002 Aerobic 138.3 9.12 15.20002 Microaerobic 48.2 5.52 8.73 0002 Anaerobic 54.7 1.35 40.5 0008nAerobic 255.8 5.37 47.6 0008n Microaerobic 127.9 3.05 41.9 0008nAnaerobic 60.8 0.62 98.1 0035 Aerobic 21.3 14.0 1.52 0035 Microaerobic13.1 4.14 3.16 0035 Anaerobic 21.3 1.06 20.1

As discussed above, it may be advantageous to use a route for converting4-HB to 4-HB-CoA that does not generate acetate as a byproduct. To thisaim, we tested the use of phosphotransbutyrylase (ptb) and butyratekinase (bk) from C. acetobutylicum to carry out this conversion viasteps 10 and 11 in FIG. 1. The native ptb/bk operon from C.acetobutylicum (genes 0020 and 0021) was cloned and expressed in pZA33.Extracts from cells containing the resulting construct were taken andassayed for the two enzyme activities as described herein. The specificactivity of BK was approximately 65 U/mg, while the specific activity ofPTB was approximately 5 U/mg. One unit (U) of activity is defined asconversion of 1 μM substrate in 1 minute at room temperature. Finally,the construct was tested for participation in the conversion of 4-HB toBDO. Host strains were transformed with the pZA33-0020-0021 constructdescribed and pZE13-0002, and compared to use of cat2 in BDO productionusing the aerobic procedure used above in FIG. 5. The BK/PTB strainproduced 1 mM BDO, compared to 2 mM when using cat2 (Table 11).Interestingly, the results were dependent on whether the host straincontained a deletion in the native adhE gene.

TABLE 11 Absolute and normalized BDO concentrations from cultures ofcells expressing adhE2 from C. acetobutylicum in pZE13 along with eithercat2 from P. gingivalis (0034) or the PTB/BK genes from C.acetobutylicum on pZA33. Host strains were either MG1655 lacI^(Q) orMG1655 ΔadhE lacI^(Q). BDO OD BDO/ Genes Host Strain (μM) (600 nm) OD0034 MG1655 lacI^(Q) 0.827 19.9 0.042 0020 + 0021 MG1655 lacI^(Q) 0.0079.8 0.0007 0034 MG1655 ΔadhE lacI^(Q) 2.084 12.5 0.166 0020 + 0021MG1655 ΔadhE lacI^(Q) 0.975 18.8 0.052

Production of BDO from Glucose.

The final step of pathway corroboration is to express both the 4-HB andBDO segments of the pathway in E. coli and demonstrate production of BDOin glucose minimal medium. New plasmids were constructed so that all therequired genes fit on two plasmids. In general, cat1, adhE, and sucDgenes were expressed from pZE13, and cat2 and 4-HBd were expressed frompZA33. Various combinations of gene source and gene order were tested inthe MG1655 lacI^(Q) background. Cells were grown anaerobically in M9minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 1.0 g/LNH₄Cl, 1 mM MgSO₄, 0.1 mM CaCl₂) supplemented with 20 g/L glucose, 100mM 3-(N-morpholino)propanesulfonic acid (MOPS) to improve the bufferingcapacity, 10 μg/mL thiamine, and the appropriate antibiotics. 0.25 mMIPTG was added approximately 15 hours following inoculation, and culturesupernatant samples taken for BDO, 4-HB, and succinate analysis 24 and48 hours following induction. The production of BDO appeared to show adependency on gene order (Table 12). The highest BDO production, over0.5 mM, was obtained with cat2 expressed first, followed by 4-HBd onpZA33, and cat1 followed by P. gingivalis sucD on pZE13. The addition ofC. acetobutylicum adhE2 in the last position on pZE13 resulted in slightimprovement. 4-HB and succinate were also produced at higherconcentrations.

TABLE 12 Production of BDO, 4-HB, and succinate in recombinant E. colistrains expressing combinations of BDO pathway genes, grown in minimalmedium supplemented with 20 g/L glucose. Concentrations are given in mM.24 Hours 48 Hours Induction OD600 OD600 Sample pZE13 pZA33 OD nm Su 4HBBDO nm Su 4HB BDO 1 cat1(0004)-sucD(0035) 4hbd (0036)-cat2(0034) 0.921.29 5.44 1.37 0.240 1.24 6.42 1.49 0.280 2 cat1(0004)-sucD(0008N) 4hbd(0036)-cat2(0034) 0.36 1.11 6.90 1.24 0.011 1.06 7.63 1.33 0.011 3adhE(0002)-cat1(0004)-sucD(0035) 4hbd (0036)-cat2(0034) 0.20 0.44 0.341.84 0.050 0.60 1.93 2.67 0.119 4 cat1(0004)-sucD(0035)-adhE(0002) 4hbd(0036)-cat2(0034) 1.31 1.90 9.02 0.73 0.073 1.95 9.73 0.82 0.077 5adhE(0002)-cat1(0004)-sucD(0008N) 4hbd (0036)-cat2(0034) 0.17 0.45 1.041.04 0.008 0.94 7.13 1.02 0.017 6 cat1(0004)-sucD(0008N)-adhE(0002) 4hbd(0036)-cat2(0034) 1.30 1.77 10.47 0.25 0.004 1.80 11.49 0.28 0.003 7cat1(0004)-sucD(0035) cat2(0034)-4hbd(0036) 1.09 1.29 5.63 2.15 0.4611.38 6.66 2.30 0.520 8 cat1(0004)-sucD(0008N) cat2(0034)-4hbd(0036) 1.812.01 11.28 0.02 0.000 2.24 11.13 0.02 0.000 9adhE(0002)-cat1(0004)-sucD(0035) cat2(0034)-4hbd(0036) 0.24 1.99 2.022.32 0.106 0.89 4.85 2.41 0.186 10 cat1(0004)-sucD(0035)-adhE(0002)cat2(0034)-4hbd(0036) 0.98 1.17 5.30 2.08 0.569 1.33 6.15 2.14 0.640 11adhE(0002)-cat1(0004)-sucD(0008N) cat2(0034)-4hbd(0036) 0.20 0.53 1.382.30 0.019 0.91 8.10 1.49 0.034 12 cat1(0004)-sucD(0008N)-adhE(0002)cat2(0034)-4hbd(0036) 2.14 2.73 12.07 0.16 0.000 3.10 11.79 0.17 0.00213 vector only vector only 2.11 2.62 9.03 0.01 0.000 3.00 12.05 0.010.000

Analysis of BDO, 4-HB and Succinate by GCMS.

BDO, 4-HB and succinate in fermentation and cell culture samples werederivatized by silylation and quantitatively analyzed by GCMS usingmethods adapted from literature reports ((Simonov et al., J. Anal Chem.59:965-971 (2004)). The developed method demonstrated good sensitivitydown to 1 μM, linearity up to at least 25 mM, as well as excellentselectivity and reproducibility.

Sample preparation was performed as follows: 100 μL filtered (0.2 μm or0.45 μm syringe filters) samples, e.g. fermentation broth, cell cultureor standard solutions, were dried down in a Speed Vac Concentrator(Savant SVC-100H) for approximately 1 hour at ambient temperature,followed by the addition of 20 μL 10 mM cyclohexanol solution, as aninternal standard, in dimethylformamide. The mixtures were vortexed andsonicated in a water bath (Branson 3510) for 15 min to ensurehomogeneity. 100 μL, silylation derivatization reagent,N,O-bis(trimethylsilyl)triflouro-acetimide (BSTFA) with 1%trimethylchlorosilane, was added, and the mixture was incubated at 70°C. for 30 min. The derivatized samples were centrifuged for 5 min, andthe clear solutions were directly injected into GCMS. All the chemicalsand reagents were from Sigma-Aldrich, with the exception of BDO whichwas purchased from J. T. Baker.

GCMS was performed on an Agilent gas chromatograph 6890N, interfaced toa mass-selective detector (MSD) 5973N operated in electron impactionization (EI) mode has been used for the analysis. A DB-5MS capillarycolumn (J&W Scientific, Agilent Technologies), 30 m×0.25 mm i.d.×0.25 μmfilm thickness, was used. The GC was operated in a split injection modeintroducing 1 μL of sample at 20:1 split ratio. The injection porttemperature was 250° C. Helium was used as a carrier gas, and the flowrate was maintained at 1.0 mL/min. A temperature gradient program wasoptimized to ensure good resolution of the analytes of interest andminimum matrix interference. The oven was initially held at 80° C. for 1min, then ramped to 120° C. at 2° C./min, followed by fast ramping to320° C. at 100° C./min and final hold for 6 min at 320° C. The MSinterface transfer line was maintained at 280° C. The data were acquiredusing ‘lowmass’ MS tune settings and 30-400 m/z mass-range scan. Thetotal analysis time was 29 min including 3 min solvent delay. Theretention times corresponded to 5.2, 10.5, 14.0 and 18.2 min forBSTFA-derivatized cyclohexanol, BDO, 4-HB and succinate, respectively.For quantitative analysis, the following specific mass fragments wereselected (extracted ion chromatograms): m/z 157 for internal standardcyclohexanol, 116 for BDO, and 147 for both 4-HB and succinate. Standardcalibration curves were constructed using analyte solutions in thecorresponding cell culture or fermentation medium to match sample matrixas close as possible. GCMS data were processed using Environmental DataAnalysis ChemStation software (Agilent Technologies).

The results indicated that most of the 4-HB and BDO produced werelabeled with ¹³C (FIG. 6, right-hand sides). Mass spectra from aparallel culture grown in unlabeled glucose are shown for comparison(FIG. 6, left-hand sides). Note that the peaks seen are for fragments ofthe derivatized molecule containing different numbers of carbon atomsfrom the metabolite. The derivatization reagent also contributes somecarbon and silicon atoms that naturally-occurring label distribution, sothe results are not strictly quantitative.

Production of BDO from 4-HB Using Alternate Pathways.

The various alternate pathways were also tested for BDO production. Thisincludes use of the native E. coli SucCD enzyme to convert succinate tosuccinyl-CoA (Table 13, rows 2-3), use of α-ketoglutarate decarboxylasein the α-ketoglutarate pathway (Table 13, row 4), and use of PTB/BK asan alternate means to generate the CoA-derivative of 4HB (Table 13, row1). Strains were constructed containing plasmids expressing the genesindicated in Table 13, which encompass these variants. The results showthat in all cases, production of 4-HB and BDO occurred (Table 13).

TABLE 13 Production of BDO, 4-HB, and succinate in recombinant E. colistrains genes for different BDO pathway variants, grown anaerobically inminimal medium supplemented with 20 g/L glucose, and harvested 24 hoursafter induction with 0.1 mM IPTG. Concentrations are given in mM. Geneson pZE13 Genes on pZA33 Succinate 4-HB BDO 0002 + 0004 + 0020n − 0021n −0.336 2.91 0.230 0035 0036 0038 + 0035  0034 − 0036 0.814 2.81 0.1260038 + 0035  0036 − 0034 0.741 2.57 0.114 0035 + 0032  0034 − 0036 5.010.538 0.154

Example III Biosynthesis of 4-Hydroxybutanoic Acid, γ-Butyrolactone and1,4-Butanediol

This Example describes the biosynthetic production of 4-hydroxybutanoicacid, γ-butyrolactone and 1,4-butanediol using fermentation and otherbioprocesses.

Methods for the integration of the 4-HB fermentation step into acomplete process for the production of purified GBL, 1,4-butanediol(BDO) and tetrahydrofuran (THF) are described below. Since 4-HB and GBLare in equilibrium, the fermentation broth will contain both compounds.At low pH this equilibrium is shifted to favor GBL. Therefore, thefermentation can operate at pH 7.5 or less, generally pH 5.5 or less.After removal of biomass, the product stream enters into a separationstep in which GBL is removed and the remaining stream enriched in 4-HBis recycled. Finally, GBL is distilled to remove any impurities. Theprocess operates in one of three ways: 1) fed-batch fermentation andbatch separation; 2) fed-batch fermentation and continuous separation;3) continuous fermentation and continuous separation. The first two ofthese modes are shown schematically in FIG. 7. The integratedfermentation procedures described below also are used for the BDOproducing cells of the invention for biosynthesis of BDO and subsequentBDO family products.

Fermentation Protocol to Produce 4-HB/GBL (Batch):

The production organism is grown in a 10 L bioreactor sparged with anN₂/CO₂ mixture, using 5 L broth containing 5 g/L potassium phosphate,2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L cornsteep liquor, and an initial glucose concentration of 20 g/L. As thecells grow and utilize the glucose, additional 70% glucose is fed intothe bioreactor at a rate approximately balancing glucose consumption.The temperature of the bioreactor is maintained at 30 degrees C. Growthcontinues for approximately 24 hours, until 4-HB reaches a concentrationof between 20-200 g/L, with the cell density being between 5 and 10 g/L.The pH is not controlled, and will typically decrease to pH 3-6 by theend of the run. Upon completion of the cultivation period, the fermentercontents are passed through a cell separation unit (e.g., centrifuge) toremove cells and cell debris, and the fermentation broth is transferredto a product separations unit. Isolation of 4-HB and/or GBL would takeplace by standard separations procedures employed in the art to separateorganic products from dilute aqueous solutions, such as liquid-liquidextraction using a water immiscible organic solvent (e.g., toluene) toprovide an organic solution of 4-HB/GBL. The resulting solution is thensubjected to standard distillation methods to remove and recycle theorganic solvent and to provide GBL (boiling point 204-205° C.) which isisolated as a purified liquid.

Fermentation Protocol to Produce 4-HB/GBL (Fully Continuous):

The production organism is first grown up in batch mode using theapparatus and medium composition described above, except that theinitial glucose concentration is 30-50 g/L. When glucose is exhausted,feed medium of the same composition is supplied continuously at a ratebetween 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate.The 4-HB concentration in the bioreactor remains constant at 30-40 g/L,and the cell density remains constant between 3-5 g/L. Temperature ismaintained at 30 degrees C., and the pH is maintained at 4.5 usingconcentrated NaOH and HCl, as required. The bioreactor is operatedcontinuously for one month, with samples taken every day to assureconsistency of 4-HB concentration. In continuous mode, fermentercontents are constantly removed as new feed medium is supplied. The exitstream, containing cells, medium, and products 4-HB and/or GBL, is thensubjected to a continuous product separations procedure, with or withoutremoving cells and cell debris, and would take place by standardcontinuous separations methods employed in the art to separate organicproducts from dilute aqueous solutions, such as continuous liquid-liquidextraction using a water immiscible organic solvent (e.g., toluene) toprovide an organic solution of 4-HB/GBL. The resulting solution issubsequently subjected to standard continuous distillation methods toremove and recycle the organic solvent and to provide GBL (boiling point204-205° C.) which is isolated as a purified liquid.

GBL Reduction Protocol:

Once GBL is isolated and purified as described above, it will then besubjected to reduction protocols such as those well known in the art(references cited) to produce 1,4-butanediol or tetrahydrofuran (THF) ora mixture thereof. Heterogeneous or homogeneous hydrogenation catalystscombined with GBL under hydrogen pressure are well known to provide theproducts 1,4-butanediol or tetrahydrofuran (THF) or a mixture thereof.It is important to note that the 4-HB/GBL product mixture that isseparated from the fermentation broth, as described above, may besubjected directly, prior to GBL isolation and purification, to thesesame reduction protocols to provide the products 1,4-butanediol ortetrahydrofuran or a mixture thereof. The resulting products,1,4-butanediol and THF are then isolated and purified by procedures wellknown in the art.

Fermentation and Hydrogenation Protocol to Produce BDO or THF Directly(Batch):

Cells are grown in a 10 L bioreactor sparged with an N₂/CO₂ mixture,using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammoniumchloride, 0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, andan initial glucose concentration of 20 g/L. As the cells grow andutilize the glucose, additional 70% glucose is fed into the bioreactorat a rate approximately balancing glucose consumption. The temperatureof the bioreactor is maintained at 30 degrees C. Growth continues forapproximately 24 hours, until 4-HB reaches a concentration of between20-200 g/L, with the cell density being between 5 and 10 g/L. The pH isnot controlled, and will typically decrease to pH 3-6 by the end of therun. Upon completion of the cultivation period, the fermenter contentsare passed through a cell separation unit (e.g., centrifuge) to removecells and cell debris, and the fermentation broth is transferred to areduction unit (e.g., hydrogenation vessel), where the mixture 4-HB/GBLis directly reduced to either 1,4-butanediol or THF or a mixturethereof. Following completion of the reduction procedure, the reactorcontents are transferred to a product separations unit. Isolation of1,4-butanediol and/or THF would take place by standard separationsprocedures employed in the art to separate organic products from diluteaqueous solutions, such as liquid-liquid extraction using a waterimmiscible organic solvent (e.g., toluene) to provide an organicsolution of 1,4-butanediol and/or THF. The resulting solution is thensubjected to standard distillation methods to remove and recycle theorganic solvent and to provide 1,4-butanediol and/or THF which areisolated as a purified liquids.

Fermentation and Hydrogenation Protocol to Produce Bdo or Thf Directly(Fully Continuous):

The cells are first grown up in batch mode using the apparatus andmedium composition described above, except that the initial glucoseconcentration is 30-50 g/L. When glucose is exhausted, feed medium ofthe same composition is supplied continuously at a rate between 0.5 L/hrand 1 L/hr, and liquid is withdrawn at the same rate. The 4-HBconcentration in the bioreactor remains constant at 30-40 g/L, and thecell density remains constant between 3-5 g/L. Temperature is maintainedat 30 degrees C., and the pH is maintained at 4.5 using concentratedNaOH and HCl, as required. The bioreactor is operated continuously forone month, with samples taken every day to assure consistency of 4-HBconcentration. In continuous mode, fermenter contents are constantlyremoved as new feed medium is supplied. The exit stream, containingcells, medium, and products 4-HB and/or GBL, is then passed through acell separation unit (e.g., centrifuge) to remove cells and cell debris,and the fermentation broth is transferred to a continuous reduction unit(e.g., hydrogenation vessel), where the mixture 4-HB/GBL is directlyreduced to either 1,4-butanediol or THF or a mixture thereof. Followingcompletion of the reduction procedure, the reactor contents aretransferred to a continuous product separations unit. Isolation of1,4-butanediol and/or THF would take place by standard continuousseparations procedures employed in the art to separate organic productsfrom dilute aqueous solutions, such as liquid-liquid extraction using awater immiscible organic solvent (e.g., toluene) to provide an organicsolution of 1,4-butanediol and/or THF. The resulting solution is thensubjected to standard continuous distillation methods to remove andrecycle the organic solvent and to provide 1,4-butanediol and/or THFwhich are isolated as a purified liquids.

Fermentation Protocol to Produce BDO Directly (Batch):

The production organism is grown in a 10 L bioreactor sparged with anN₂/CO₂ mixture, using 5 L broth containing 5 g/L potassium phosphate,2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L cornsteep liquor, and an initial glucose concentration of 20 g/L. As thecells grow and utilize the glucose, additional 70% glucose is fed intothe bioreactor at a rate approximately balancing glucose consumption.The temperature of the bioreactor is maintained at 30 degrees C. Growthcontinues for approximately 24 hours, until BDO reaches a concentrationof between 20-200 g/L, with the cell density generally being between 5and 10 g/L. Upon completion of the cultivation period, the fermentercontents are passed through a cell separation unit (e.g., centrifuge) toremove cells and cell debris, and the fermentation broth is transferredto a product separations unit. Isolation of BDO would take place bystandard separations procedures employed in the art to separate organicproducts from dilute aqueous solutions, such as liquid-liquid extractionusing a water immiscible organic solvent (e.g., toluene) to provide anorganic solution of BDO. The resulting solution is then subjected tostandard distillation methods to remove and recycle the organic solventand to provide BDO (boiling point 228-229° C.) which is isolated as apurified liquid.

Fermentation Protocol to Produce BDO Directly (Fully Continuous):

The production organism is first grown up in batch mode using theapparatus and medium composition described above, except that theinitial glucose concentration is 30-50 g/L. When glucose is exhausted,feed medium of the same composition is supplied continuously at a ratebetween 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate.The BDO concentration in the bioreactor remains constant at 30-40 g/L,and the cell density remains constant between 3-5 g/L. Temperature ismaintained at 30 degrees C., and the pH is maintained at 4.5 usingconcentrated NaOH and HCl, as required. The bioreactor is operatedcontinuously for one month, with samples taken every day to assureconsistency of BDO concentration. In continuous mode, fermenter contentsare constantly removed as new feed medium is supplied. The exit stream,containing cells, medium, and the product BDO, is then subjected to acontinuous product separations procedure, with or without removing cellsand cell debris, and would take place by standard continuous separationsmethods employed in the art to separate organic products from diluteaqueous solutions, such as continuous liquid-liquid extraction using awater immiscible organic solvent (e.g., toluene) to provide an organicsolution of BDO. The resulting solution is subsequently subjected tostandard continuous distillation methods to remove and recycle theorganic solvent and to provide BDO (boiling point 228-229° C.) which isisolated as a purified liquid (mpt 20° C.).

Example IV Exemplary BDO Pathways

This example describes exemplary enzymes and corresponding genes for1,4-butandiol (BDO) synthetic pathways.

Exemplary BDO synthetic pathways are shown in FIGS. 8-13. The pathwaysdepicted in FIGS. 8-13 are from common central metabolic intermediatesto 1,4-butanediol. All transformations depicted in FIGS. 8-13 fall intothe 18 general categories of transformations shown in Table 14. Below isdescribed a number of biochemically characterized candidate genes ineach category. Specifically listed are genes that can be applied tocatalyze the appropriate transformations in FIGS. 9-13 when cloned andexpressed in a host organism. The top three exemplary genes for each ofthe key steps in FIGS. 9-13 are provided in Tables 15-23 (see below).Exemplary genes were provided for the pathways depicted in FIG. 8 aredescribed herein.

TABLE 14 Enzyme types required to convert common central metabolicintermediates into 1,4-butanediol. The first three digits of each labelcorrespond to the first three Enzyme Commission number digits whichdenote the general type of transformation independent of substratespecificity. Label Function 1.1.1.a Oxidoreductase (ketone to hydroxylor aldehyde to alcohol) 1.1.1.c Oxidoreductase (2 step, acyl-CoA toalcohol) 1.2.1.b Oxidoreductase (acyl-CoA to aldehyde) 1.2.1.cOxidoreductase (2-oxo acid to acyl-CoA, decarboxylation) 1.2.1.dOxidoreductase (phosphorylating/dephosphorylating) 1.3.1.aOxidoreductase operating on CH—CH donors 1.4.1.a Oxidoreductaseoperating on amino acids 2.3.1.a Acyltransferase (transferring phosphategroup) 2.6.1.a Aminotransferase 2.7.2.a Phosphotransferase, carboxylgroup acceptor 2.8.3.a Coenzyme-A transferase 3.1.2.a Thiolesterhydrolase (CoA specific) 4.1.1.a Carboxy-lyase 4.2.1.a Hydro-lyase4.3.1.a Ammonia-lyase 5.3.3.a Isomerase 5.4.3.a Aminomutase 6.2.1.aAcid-thiol ligase1.1.1.a—Oxidoreductase (Aldehyde to Alcohol or Ketone to Hydroxyl)

Aldehyde to Alcohol.

Exemplary genes encoding enzymes that catalyze the conversion of analdehyde to alcohol, that is, alcohol dehydrogenase or equivalentlyaldehyde reductase, include alrA encoding a medium-chain alcoholdehydrogenase for C2-C14 (Tani et al. Appl. Environ. Microbiol.66:5231-5235 (2000)), ADH2 from Saccharomyces cerevisiae (Atsumi et al.Nature 451:86-89 (2008)), yqhD from E. coli which has preference formolecules longer than C(3) (Sulzenbacher et al. Journal of MolecularBiology 342:489-502 (2004)), and bdh I and bdh II from C. acetobutylicumwhich converts butyraldehyde into butanol (Walter et al. Journal ofBacteriology 174:7149-7158 (1992)). The protein sequences for each ofthese exemplary gene products, if available, can be found using thefollowing GenBank accession numbers:

Gene Accession No. GI No. Organism alrA BAB12273.1 9967138 Acinetobactersp. Strain M-1 ADH2 NP_014032.1 6323961 Saccharymyces cerevisiae yqhDNP_417484.1 16130909 Escherichia coli bdh I NP_349892.1 15896543Clostridium acetobutylicum bdh II NP_349891.1 15896542 Clostridiumacetobutylicum

Enzymes exhibiting 4-hydroxybutyrate dehydrogenase activity (EC1.1.1.61) also fall into this category. Such enzymes have beencharacterized in Ralstonia eutropha (Bravo et al. J. Forensic Sci.49:379-387 (2004), Clostridium kluyveri (Wolff et al. Protein Expr.Purif. 6:206-212 (1995)) and Arabidopsis thaliana (Breitkreuz et al. J.Biol. Chem. 278:41552-41556 (2003)).

Gene Accession No. GI No. Organism 4hbd YP_726053.1 113867564 Ralstoniaeutropha H16 4hbd EDK35022.1 146348486 Clostridium kluyveri DSM 555 4hbdQ94B07 75249805 Arabidopsis thaliana

Another exemplary enzyme is 3-hydroxyisobutyrate dehydrogenase whichcatalyzes the reversible oxidation of 3-hydroxyisobutyrate tomethylmalonate semialdehyde. This enzyme participates in valine, leucineand isoleucine degradation and has been identified in bacteria,eukaryotes, and mammals. The enzyme encoded by P84067 from Thermusthermophilus HB8 has been structurally characterized (Lokanath et al. JMol Biol 352:905-17 (2005)). The reversibility of the human3-hydroxyisobutyrate dehydrogenase was demonstrated usingisotopically-labeled substrate (Manning et al. Biochem J 231:481-484(1985)). Additional genes encoding this enzyme include 3hidh in Homosapiens (Hawes et al. Methods Enzymol. 324:218-228 (2000)) andOryctolagus cuniculus (Chowdhury et al. Biosci. Biotechnol Biochem.60:2043-2047 (1996); Hawes et al. Methods Enzymol. 324:218-228 (2000)),mmsb in Pseudomonas aeruginosa, and dhat in Pseudomonas putida (Aberhartet al. J Chem. Soc. [Perkin 1] 6:1404-1406 (1979); Chowdhury et al.Biosci. Biotechnol Biochem. 67:438-441 (2003); Chowdhury et al. Biosci.Biotechnol Biochem. 60:2043-2047 (1996)).

Gene Accession No. GI No. Organism P84067 P84067 75345323 Thermusthermophilus mmsb P28811.1 127211 Pseudomonas aeruginosa dhat Q59477.12842618 Pseudomonas putida 3hidh P31937.2 12643395 Homo sapiens 3hidhP32185.1 416872 Oryctolagus cuniculus

Several 3-hydroxyisobutyrate dehydrogenase enzymes have also been shownto convert malonic semialdehyde to 3-hydroxyproprionic acid (3-HP).Three gene candidates exhibiting this activity are mmsB from Pseudomonasaeruginosa PAO1(62), mmsB from Pseudomonas putida KT2440 (Liao et al.,US Publication 2005/0221466) and mmsB from Pseudomonas putida E23(Chowdhury et al., Biosci. Biotechnol. Biochem. 60:2043-2047 (1996)). Anenzyme with 3-hydroxybutyrate dehydrogenase activity in Alcaligenesfaecalis M3A has also been identified (Gokam et al., U.S. Pat. No.7,393,676; Liao et al., US Publication No. 2005/0221466). Additionalgene candidates from other organisms including Rhodobacter spaeroidescan be inferred by sequence similarity.

Gene Accession No. GI No. Organism mmsB AAA25892.1 151363 Pseudomonasaeruginosa mmsB NP_252259.1 15598765 Pseudomonas aeruginosa PAO1 mmsBNP_746775.1 26991350 Pseudomonas putida KT2440 mmsB JC7926 60729613Pseudomonas putida E23 orfB1 AAL26884 16588720 Rhodobacter spaeroides

The conversion of malonic semialdehyde to 3-HP can also be accomplishedby two other enzymes: NADH-dependent 3-hydroxypropionate dehydrogenaseand NADPH-dependent malonate semialdehyde reductase. An NADH-dependent3-hydroxypropionate dehydrogenase is thought to participate inbeta-alanine biosynthesis pathways from propionate in bacteria andplants (Rathinasabapathi, B. Journal of Plant Pathology 159:671-674(2002); Stadtman, E. R. J. Am. Chem. Soc. 77:5765-5766 (1955)). Thisenzyme has not been associated with a gene in any organism to date.NADPH-dependent malonate semialdehyde reductase catalyzes the reversereaction in autotrophic CO₂-fixing bacteria. Although the enzymeactivity has been detected in Metallosphaera sedula, the identity of thegene is not known (Alber et al. J. Bacteriol. 188:8551-8559 (2006)).

Ketone to Hydroxyl.

There exist several exemplary alcohol dehydrogenases that convert aketone to a hydroxyl functional group. Two such enzymes from E. coli areencoded by malate dehydrogenase (mdh) and lactate dehydrogenase (ldhA).In addition, lactate dehydrogenase from Ralstonia eutropha has beenshown to demonstrate high activities on substrates of various chainlengths such as lactate, 2-oxobutyrate, 2-oxopentanoate and2-oxoglutarate (Steinbuchel, A. and H. G. Schlegel Eur. J. Biochem.130:329-334 (1983)). Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al. Biochem.Biophys. Res. Commun. 77:586-591 (1977)). An additional candidate forthis step is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh)from the human heart which has been cloned and characterized (Marks etal. J. Biol. Chem. 267:15459-15463 (1992)). This enzyme is adehydrogenase that operates on a 3-hydroxyacid. Another exemplaryalcohol dehydrogenase converts acetone to isopropanol as was shown in C.beijerinckii (Ismaiel et al. J. Bacteriol. 175:5097-5105 (1993)) and T.brockii (Lamed et al. Biochem. J. 195:183-190 (1981); Peretz andBurstein Biochemistry 28:6549-6555 (1989)).

Gene Accession No. GI No. Organism mdh AAC76268.1 1789632 Escherichiacoli ldhA NP_415898.1 16129341 Escherichia coli ldh YP_725182.1113866693 Ralstonia eutropha bdh AAA58352.1 177198 Homo sapiens adhAAA23199.2 60592974 Clostridium beijerinckii NRRL B593 adh P14941.1113443 Thermoanaerobacter brockii HTD4

Exemplary 3-hydroxyacyl dehydrogenases which convert acetoacetyl-CoA to3-hydroxybutyryl-CoA include hbd from C. acetobutylicum (Boynton et al.Journal of Bacteriology 178:3015-3024 (1996)), hbd from C. beijerinckii(Colby et al. Appl Environ. Microbiol 58:3297-3302 (1992)), and a numberof similar enzymes from Metallosphaera sedula (Berg et al. Archaea.Science. 318:1782-1786 (2007)).

Gene Accession No. GI No. Organism hbd NP_349314.1 15895965 Clostridiumacetobutylicum hbd AAM14586.1 20162442 Clostridium beijerinckiiMsed_1423 YP_001191505 146304189 Metallosphaera sedula Msed_0399YP_001190500 146303184 Metallosphaera sedula Msed_0389 YP_001190490146303174 Metallosphaera sedula Msed_1993 YP_001192057 146304741Metallosphaera sedula1.1.1.c—Oxidoredutase (2 Step, Acyl-CoA to Alcohol)

Exemplary 2-step oxidoreductases that convert an acyl-CoA to alcoholinclude those that transform substrates such as acetyl-CoA to ethanol(for example, adhE from E. coli (Kessler et al. FEBS. Lett. 281:59-63(1991)) and butyryl-CoA to butanol (for example, adhE2 from C.acetobutylicum (Fontaine et al. J. Bacteriol. 184:821-830 (2002)). Inaddition to reducing acetyl-CoA to ethanol, the enzyme encoded by adhEin Leuconostoc mesenteroides has been shown to oxide the branched chaincompound isobutyraldehyde to isobutyryl-CoA (Kazahaya et al. J. Gen.Appl. Microbiol. 18:43-55 (1972); Koo et al. Biotechnol Lett. 27:505-510(2005)).

Gene Accession No. GI No. Organism adhE NP _415757.1 16129202Escherichia coli adhE2 AAK09379.1 12958626 Clostridium acetobutylicumadhE AAV66076.1 AAV66076 Leuconostoc mesenteroides

Another exemplary enzyme can convert malonyl-CoA to 3-HP. AnNADPH-dependent enzyme with this activity has characterized inChloroflexus aurantiacus where it participates in the3-hydroxypropionate cycle (Hugler et al., J. Bacteriol. 184:2404-2410(2002); Strauss and Fuchs, Eur. J. Biochem. 215:633-643 (1993)). Thisenzyme, with a mass of 300 kDa, is highly substrate-specific and showslittle sequence similarity to other known oxidoreductases (Hugler etal., J. Bacteriol. 184:2404-2410 (2002)). No enzymes in other organismshave been shown to catalyze this specific reaction; however there isbioinformatic evidence that other organisms may have similar pathways(Klatt et al., Environ. Microbiol. 9:2067-2078 (2007)). Enzymecandidates in other organisms including Roseiflexus castenholzii,Erythrobacter sp. NAP1 and marine gamma proteobacterium HTCC2080 can beinferred by sequence similarity.

Gene Accession No. GI No. Organism mcr AAS20429.1 42561982 Chloroflexusaurantiacus Rcas_2929 YP_001433009.1 156742880 Roseiflexus castenholziiNAP1_02720 ZP_01039179.1 85708113 Erythrobacter sp. NAP1 MGP2080_00535ZP_01626393.1 119504313 marine gamma proteobacterium HTCC2080

Longer chain acyl-CoA molecules can be reduced by enzymes such as thejojoba (Simmondsia chinensis) FAR which encodes an alcohol-forming fattyacyl-CoA reductase. Its overexpression in E. coli resulted in FARactivity and the accumulation of fatty alcohol (Metz et al. PlantPhysiology 122:635-644) 2000)).

Gene Accession No. GI No. Organism FAR AAD38039.1 5020215 Simmondsiachinensis1.2.1.b—Oxidoreductase (Acyl-CoA to Aldehyde)

Several acyl-CoA dehydrogenases are capable of reducing an acyl-CoA toits corresponding aldehyde. Exemplary genes that encode such enzymesinclude the Acinetobacter calcoaceticus arc1 encoding a fatty acyl-CoAreductase (Reiser and Somerville, J. Bacteriology 179:2969-2975 (1997)),the Acinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al. Appl.Environ. Microbiol. 68:1192-1195 (2002)), and a CoA- and NADP-dependentsuccinate semialdehyde dehydrogenase encoded by the sucD gene inClostridium kluyveri (Sohling and Gottschalk J Bacteriol 178:871-80(1996); Sohling and Gottschalk J Bacteriol. 178:871-880 (1996)). SucD ofP. gingivalis is another succinate semialdehyde dehydrogenase (Takahashiet al. J. Bacteriol. 182:4704-4710 (2000)). The enzyme acylatingacetaldehyde dehydrogenase in Pseudomonas sp, encoded by bphG, is yetanother as it has been demonstrated to oxidize and acylate acetaldehyde,propionaldehyde, butyraldehyde, isobutyraldehyde and formaldehyde(Powlowski et al. J Bacteriol. 175:377-385 (1993)).

Gene Accession No. GI No. Organism acr1 YP_047869.1 50086359Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyiacr1 BAB85476.1 18857901 Acinetobacter sp. Strain M-1 sucD P38947.1730847 Clostridium kluyveri sucD NP_904963.1 34540484 Porphyromonasgingivalis bphG BAA03892.1 425213 Pseudomonas sp

An additional enzyme type that converts an acyl-CoA to its correspondingaldehyde is malonyl-CoA reductase which transforms malonyl-CoA tomalonic semialdehyde. Malonyl-CoA reductase is a key enzyme inautotrophic carbon fixation via the 3-hydroxypropionate cycle inthermoacidophilic archael bacteria (Berg et al. Science 318:1782-1786(2007); Thauer, R. K. Science 318:1732-1733 (2007)). The enzyme utilizesNADPH as a cofactor and has been characterized in Metallosphaera andSulfolobus spp (Alber et al. J. Bacteriol. 188:8551-8559 (2006); Hugleret al. J. Bacteriol. 184:2404-2410 (2002)). The enzyme is encoded byMsed_(—)0709 in Metallosphaera sedula (Alber et al. J. Bacteriol.188:8551-8559 (2006); Berg et al. Science 318:1782-1786 (2007)). A geneencoding a malonyl-CoA reductase from Sulfolobus tokodaii was cloned andheterologously expressed in E. coli (Alber et al. J. Bacteriol.188:8551-8559 (2006)). Although the aldehyde dehydrogenase functionalityof these enzymes is similar to the bifunctional dehydrogenase fromChloroflexus aurantiacus, there is little sequence similarity. Bothmalonyl-CoA reductase enzyme candidates have high sequence similarity toaspartate-semialdehyde dehydrogenase, an enzyme catalyzing the reductionand concurrent dephosphorylation of aspartyl-4-phosphate to aspartatesemialdehyde. Additional gene candidates can be found by sequencehomology to proteins in other organisms including Sulfolobussolfataricus and Sulfolobus acidocaldarius.

Gene Accession No. GI No. Organism Msed_0709 YP_001190808.1 146303492Metallosphaera sedula mcr NP_378167.1 15922498 Sulfolobus tokodaii asd-2NP_343563.1 15898958 Sulfolobus solfataricus Saci_2370 YP_256941.170608071 Sulfolobus acidocaldarius1.2.1.c—Oxidoreductase (2-Oxo Acid to Acyl-CoA, Decarboxylation)

Enzymes in this family include 1) branched-chain 2-keto-aciddehydrogenase, 2) alpha-ketoglutarate dehydrogenase, and 3) the pyruvatedehydrogenase multienzyme complex (PDHC). These enzymes are multi-enzymecomplexes that catalyze a series of partial reactions which result inacylating oxidative decarboxylation of 2-keto-acids. Each of the2-keto-acid dehydrogenase complexes occupies key positions inintermediary metabolism, and enzyme activity is typically tightlyregulated (Fries et al. Biochemistry 42:6996-7002 (2003)). The enzymesshare a complex but common structure composed of multiple copies ofthree catalytic components: alpha-ketoacid decarboxylase (E1),dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase(E3). The E3 component is shared among all 2-keto-acid dehydrogenasecomplexes in an organism, while the E1 and E2 components are encoded bydifferent genes. The enzyme components are present in numerous copies inthe complex and utilize multiple cofactors to catalyze a directedsequence of reactions via substrate channeling. The overall size ofthese dehydrogenase complexes is very large, with molecular massesbetween 4 and 10 million Da (that is, larger than a ribosome).

Activity of enzymes in the 2-keto-acid dehydrogenase family is normallylow or limited under anaerobic conditions in E. coli. Increasedproduction of NADH (or NADPH) could lead to a redox-imbalance, and NADHitself serves as an inhibitor to enzyme function. Engineering effortshave increased the anaerobic activity of the E. coli pyruvatedehydrogenase complex (Kim et al. Appl. Environ. Microbiol. 73:1766-1771(2007); Kim et al. J. Bacteriol. 190:3851-3858) 2008); Zhou et al.Biotechnol. Lett. 30:335-342 (2008)). For example, the inhibitory effectof NADH can be overcome by engineering an H322Y mutation in the E3component (Kim et al. J. Bacteriol. 190:3851-3858 (2008)). Structuralstudies of individual components and how they work together in complexprovide insight into the catalytic mechanisms and architecture ofenzymes in this family (Aevarsson et al. Nat. Struct. Biol. 6:785-792(1999); Zhou et al. Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807(2001)). The substrate specificity of the dehydrogenase complexes variesin different organisms, but generally branched-chain keto-aciddehydrogenases have the broadest substrate range.

Alpha-ketoglutarate dehydrogenase (AKGD) converts alpha-ketoglutarate tosuccinyl-CoA and is the primary site of control of metabolic fluxthrough the TCA cycle (Hansford, R. G. Curr. Top. Bioenerg. 10:217-278(1980)). Encoded by genes sucA, sucB and lpd in E. coli, AKGD geneexpression is downregulated under anaerobic conditions and during growthon glucose (Park et al. Mol. Microbiol. 15:473-482 (1995)). Although thesubstrate range of AKGD is narrow, structural studies of the catalyticcore of the E2 component pinpoint specific residues responsible forsubstrate specificity (Knapp et al. J. Mol. Biol. 280:655-668 (1998)).The Bacillus subtilis AKGD, encoded by odhAB (E1 and E2) and pdhD (E3,shared domain), is regulated at the transcriptional level and isdependent on the carbon source and growth phase of the organism(Resnekov et al. Mol. Gen. Genet. 234:285-296 (1992)). In yeast, theLPD1 gene encoding the E3 component is regulated at the transcriptionallevel by glucose (Roy and Dawes J. Gen. Microbiol. 133:925-933 (1987)).The E1 component, encoded by KGD1, is also regulated by glucose andactivated by the products of HAP2 and HAP3 (Repetto and Tzagoloff Mol.Cell Biol. 9:2695-2705 (1989)). The AKGD enzyme complex, inhibited byproducts NADH and succinyl-CoA, is well-studied in mammalian systems, asimpaired function of has been linked to several neurological diseases(Tretter and dam-Vizi Philos. Trans. R. Soc. Lond B Biol. Sci.360:2335-2345 (2005)).

Gene Accession No. GI No. Organism sucA NP_415254.1 16128701 Escherichiacoli str. K12 substr. MG1655 sucB NP_415255.1 16128702 Escherichia colistr. K12 substr. MG1655 Lpd NP_414658.1 16128109 Escherichia coli str.K12 substr. MG1655 odhA P23129.2 51704265 Bacillus subtilis odhBP16263.1 129041 Bacillus subtilis pdhD P21880.1 118672 Bacillus subtilisKGD1 NP_012141.1 6322066 Saccharomyces cerevisiae KGD2 NP_010432.16320352 Saccharomyces cerevisiae LPD1 NP_116635.1 14318501 Saccharomycescerevisiae

Branched-chain 2-keto-acid dehydrogenase complex (BCKAD), also known as2-oxoisovalerate dehydrogenase, participates in branched-chain aminoacid degradation pathways, converting 2-keto acids derivatives ofvaline, leucine and isoleucine to their acyl-CoA derivatives and CO₂.The complex has been studied in many organisms including Bacillussubtilis (Wang et al. Eur. J. Biochem. 213:1091-1099 (1993)), Rattusnorvegicus (Namba et al. J. Biol. Chem. 244:4437-4447 (1969)) andPseudomonas putida (Sokatch J. Bacteriol. 148:647-652 (1981)). InBacillus subtilis the enzyme is encoded by genes pdhD (E3 component),bfmBB (E2 component), bfmBAA and bfmBAB (E1 component) (Wang et al. Eur.J. Biochem. 213:1091-1099 (1993)). In mammals, the complex is regulatedby phosphorylation by specific phosphatases and protein kinases. Thecomplex has been studied in rat hepatocites (Chicco et al. J. Biol.Chem. 269:19427-19434 (1994)) and is encoded by genes Bckdha (E1 alpha),Bckdhb (E1 beta), Dbt (E2), and Dld (E3). The E1 and E3 components ofthe Pseudomonas putida BCKAD complex have been crystallized (Aevarssonet al. Nat. Struct. Biol. 6:785-792 (1999); Mattevi Science255:1544-1550 (1992)) and the enzyme complex has been studied (Sokatchet al. J. Bacteriol. 148:647-652 (1981)). Transcription of the P. putidaBCKAD genes is activated by the gene product of bkdR (Hester et al. Eur.J. Biochem. 233:828-836 (1995)). In some organisms including Rattusnorvegicus (Paxton et al. Biochem. J. 234:295-303 (1986)) andSaccharomyces cerevisiae (Sinclair et al. Biochem. Mol. Biol. Int.31:911-922 (1993)), this complex has been shown to have a broadsubstrate range that includes linear oxo-acids such as 2-oxobutanoateand alpha-ketoglutarate, in addition to the branched-chain amino acidprecursors. The active site of the bovine BCKAD was engineered to favoralternate substrate acetyl-CoA (Meng and Chuang, Biochemistry33:12879-12885 (1994)).

Gene Accession No. GI No. Organism bfmBB NP_390283.1 16079459 Bacillussubtilis bfmBAA NP_390285.1 16079461 Bacillus subtilis bfmBABNP_390284.1 16079460 Bacillus subtilis pdhD P21880.1 118672 Bacillussubtilis lpdV P09063.1 118677 Pseudomonas putida bkdB P09062.1 129044Pseudomonas putida bkdA1 NP_746515.1 26991090 Pseudomonas putida bkdA2NP_746516.1 26991091 Pseudomonas putida Bckdha NP_036914.1 77736548Rattus norvegicus Bckdhb NP_062140.1 158749538 Rattus norvegicus DbtNP_445764.1 158749632 Rattus norvegicus Dld NP_955417.1 40786469 Rattusnorvegicus

The pyruvate dehydrogenase complex, catalyzing the conversion ofpyruvate to acetyl-CoA, has also been extensively studied. In the E.coli enzyme, specific residues in the E1 component are responsible forsubstrate specificity (Bisswanger, H. J Biol Chem. 256:815-822 (1981);Bremer, J. Eur. J Biochem. 8:535-540 (1969); Gong et al. J Biol Chem.275:13645-13653 (2000)). As mentioned previously, enzyme engineeringefforts have improved the E. coli PDH enzyme activity under anaerobicconditions (Kim et al. Appl. Environ. Microbiol. 73:1766-1771 (2007);Kim J. Bacteriol. 190:3851-3858 (2008); Zhou et al. Biotechnol. Lett.30:335-342 (2008)). In contrast to the E. coli PDH, the B. subtiliscomplex is active and required for growth under anaerobic conditions(Nakano J. Bacteriol. 179:6749-6755 (1997)). The Klebsiella pneumoniaePDH, characterized during growth on glycerol, is also active underanaerobic conditions (Menzel et al. J. Biotechnol. 56:135-142 (1997)).Crystal structures of the enzyme complex from bovine kidney (Zhou et al.Proc. Natl. Acad. Sci. U.S.A. 98:14802-14807 (2001)) and the E2catalytic domain from Azotobacter vinelandii are available (Mattevi etal. Science 255:1544-1550 (1992)). Some mammalian PDH enzymes complexescan react on alternate substrates such as 2-oxobutanoate, althoughcomparative kinetics of Rattus norvegicus PDH and BCKAD indicate thatBCKAD has higher activity on 2-oxobutanoate as a substrate (Paxton etal. Biochem. J. 234:295-303 (1986)).

Gene Accession No. GI No. Organism aceE NP_414656.1 16128107 Escherichiacoli str. K12 substr. MG1655 aceF NP_414657.1 16128108 Escherichia colistr. K12 substr. MG1655 lpd NP_414658.1 16128109 Escherichia coli str.K12 substr. MG1655 pdhA P21881.1 3123238 Bacillus subtilis pdhB P21882.1129068 Bacillus subtilis pdhC P21883.2 129054 Bacillus subtilis pdhDP21880.1 118672 Bacillus subtilis aceE YP_001333808.1 152968699Klebsiella pneumonia MGH78578 aceF YP_001333809.1 152968700 Klebsiellapneumonia MGH78578 lpdA YP_001333810.1 152968701 Klebsiella pneumoniaMGH78578 Pdha1 NP_001004072.2 124430510 Rattus norvegicus Pdha2NP_446446.1 16758900 Rattus norvegicus Dlat NP_112287.1 78365255 Rattusnorvegicus Dld NP_955417.1 40786469 Rattus norvegicus

As an alternative to the large multienzyme 2-keto-acid dehydrogenasecomplexes described above, some anaerobic organisms utilize enzymes inthe 2-ketoacid oxidoreductase family (OFOR) to catalyze acylatingoxidative decarboxylation of 2-keto-acids. Unlike the dehydrogenasecomplexes, these enzymes contain iron-sulfur clusters, utilize differentcofactors, and use ferredoxin or flavodixin as electron acceptors inlieu of NAD(P)H. While most enzymes in this family are specific topyruvate as a substrate (POR) some 2-keto-acid:ferredoxinoxidoreductases have been shown to accept a broad range of 2-ketoacidsas substrates including alpha-ketoglutarate and 2-oxobutanoate (Fukudaand Wakagi Biochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J.Biochem. 120:587-599 (1996)). One such enzyme is the OFOR from thethermoacidophilic archaeon Sulfolobus tokodaii 7, which contains analpha and beta subunit encoded by gene ST2300 (Fukuda and WakagiBiochim. Biophys. Acta 1597:74-80 (2002); Zhang et al. J. Biochem.120:587-599 (1996)). A plasmid-based expression system has beendeveloped for efficiently expressing this protein in E. coli (Fukuda etal. Eur. J. Biochem. 268:5639-5646 (2001)) and residues involved insubstrate specificity were determined (Fukuda and Wakagi Biochim.Biophys. Acta 1597:74-80 (2002)). Two OFORs from Aeropyrum pernix str.K1 have also been recently cloned into E. coli, characterized, and foundto react with a broad range of 2-oxoacids (Nishizawa et al. FEBS Lett.579:2319-2322 (2005)). The gene sequences of these OFOR candidates areavailable, although they do not have GenBank identifiers assigned todate. There is bioinformatic evidence that similar enzymes are presentin all archaea, some anaerobic bacteria and amitochondrial eukarya(Fukuda and Wakagi Biochim. Biophys. Acta 1597:74-80 (2005)). This classof enzyme is also interesting from an energetic standpoint, as reducedferredoxin could be used to generate NADH by ferredoxin-NAD reductase(Petitdemange et al. Biochim. Biophys. Acta 421:334-337 (1976)). Also,since most of the enzymes are designed to operate under anaerobicconditions, less enzyme engineering may be required relative to enzymesin the 2-keto-acid dehydrogenase complex family for activity in ananaerobic environment.

Gene Accession No. GI No. Organism ST2300 NP_378302.1 15922633Sulfolobus tokodaii 71.2.1.d—Oxidoreductase (Phosphorylating/Dephosphorylating)

Exemplary enzymes in this class include glyceraldehyde 3-phosphatedehydrogenase which converts glyceraldehyde-3-phosphate into D-glycerate1,3-bisphosphate (for example, E. coli gapA (Branlant and Branlant Eur.J. Biochem. 150:61-66(1985)), aspartate-semialdehyde dehydrogenase whichconverts L-aspartate-4-semialdehyde into L-4-aspartyl-phosphate (forexample, E. coli asd (Biellmann et al. Eur. J. Biochem. 104:53-58(1980)), N-acetyl-gamma-glutamyl-phosphate reductase which convertsN-acetyl-L-glutamate-5-semialdehyde into N-acetyl-L-glutamyl-5-phosphate(for example, E. coli argC (Parsot et al. Gene 68:275-283 (1988)), andglutamate-5-semialdehyde dehydrogenase which convertsL-glutamate-5-semialdehyde into L-glutamyl-5-phospate (for example, E.coli proA (Smith et al. J. Bacteriol. 157:545-551 (1984)).

Gene Accession No. GI No. Organism gapA P0A9B2.2 71159358 Escherichiacoli asd NP_417891.1 16131307 Escherichia coli argC NP_418393.1 16131796Escherichia coli proA NP_414778.1 16128229 Escherichia coli1.3.1.a—Oxidoreductase Operating on CH—CH Donors

An exemplary enoyl-CoA reductase is the gene product of bcd from C.acetobutylicum (Atsumi et al. Metab Eng (2007); Boynton et al. Journalof Bacteriology 178:3015-3024 (1996), which naturally catalyzes thereduction of crotonyl-CoA to butyryl-CoA. Activity of this enzyme can beenhanced by expressing bcd in conjunction with expression of the C.acetobutylicum etfAB genes, which encode an electron transferflavoprotein. An additional candidate for the enoyl-CoA reductase stepis the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeisteret al. Journal of Biological Chemistry 280:4329-4338 (2005)). Aconstruct derived from this sequence following the removal of itsmitochondrial targeting leader sequence was cloned in E. coli resultingin an active enzyme (Hoffmeister et al., supra, (2005)). This approachis well known to those skilled in the art of expressing eukaryoticgenes, particularly those with leader sequences that may target the geneproduct to a specific intracellular compartment, in prokaryoticorganisms. A close homolog of this gene, TDE0597, from the prokaryoteTreponema denticola represents a third enoyl-CoA reductase which hasbeen cloned and expressed in E. coli (Tucci and Martin FEBS Letters581:1561-1566 (2007)).

Gene Accession No. GI No. Organism bcd NP_349317.1 15895968 Clostridiumacetobutylicum etfA NP_349315.1 15895966 Clostridium acetobutylicum etfBNP_349316.1 15895967 Clostridium acetobutylicum TER Q5EU90.1 62287512Euglena gracilis TDE0597 NP_971211.1 42526113 Treponema denticola

Exemplary 2-enoate reductase (EC 1.3.1.31) enzymes are known to catalyzethe NADH-dependent reduction of a wide variety of α, β-unsaturatedcarboxylic acids and aldehydes (Rohdich et al. J. Biol. Chem.276:5779-5787 (2001)). 2-Enoate reductase is encoded by enr in severalspecies of Clostridia (Giesel and Simon Arch Microbiol. 135(1): p. 51-57(2001) including C. tyrobutyricum, and C. thermoaceticum (now calledMoorella thermoaceticum) (Rohdich et al., supra, (2001)). In therecently published genome sequence of C. kluyveri, 9 coding sequencesfor enoate reductases have been reported, out of which one has beencharacterized (Seedorf et al. Proc Natl Acad Sci U.S.A 105(6):2128-33(2008)). The enr genes from both C. tyrobutyricum and C. thermoaceticumhave been cloned and sequenced and show 59% identity to each other. Theformer gene is also found to have approximately 75% similarity to thecharacterized gene in C. kluyveri (Giesel and Simon Arch Microbiol135(1):51-57 (1983)). It has been reported based on these sequenceresults that enr is very similar to the dienoyl CoA reductase in E. coli(fadH) (163 Rohdich et al., supra (2001)). The C. thermoaceticum enrgene has also been expressed in an enzymatically active form in E. coli(163 Rohdich et al., supra (2001)).

Gene Accession No. GI No. Organism fadH NP_417552.1 16130976 Escherichiacoli enr ACA54153.1 169405742 Clostridium botulinum A3 str enrCAA71086.1 2765041 Clostridium tyrobutyricum enr CAA76083.1 3402834Clostridium kluyveri enr YP_430895.1 83590886 Moorella thermoacetica1.4.1.a—Oxidoreductase Operating on Amino Acids

Most oxidoreductases operating on amino acids catalyze the oxidativedeamination of alpha-amino acids with NAD+ or NADP+ as acceptor.Exemplary oxidoreductases operating on amino acids include glutamatedehydrogenase (deaminating), encoded by gdhA, leucine dehydrogenase(deaminating), encoded by ldh, and aspartate dehydrogenase(deaminating), encoded by nadX. The gdhA gene product from Escherichiacoli (Korber et al. J. Mol. Biol. 234:1270-1273 (1993); McPherson andWootton Nucleic. Acids Res. 11:5257-5266 (1983)), gdh from Thermotogamaritima (Kort et al. Extremophiles 1:52-60 (1997); Lebbink, et al. J.Mol. Biol. 280:287-296 (1998)); Lebbink et al. J. Mol. Biol. 289:357-369(1999)), and gdhA1 from Halobacterium salinarum (Ingoldsby et al. Gene349:237-244 (2005)) catalyze the reversible interconversion of glutamateto 2-oxoglutarate and ammonia, while favoring NADP(H), NAD(H), or both,respectively. The ldh gene of Bacillus cereus encodes the LeuDH proteinthat has a wide of range of substrates including leucine, isoleucine,valine, and 2-aminobutanoate (Ansorge and Kula Biotechnol Bioeng.68:557-562 (2000); Stoyan et al. J. Biotechnol 54:77-80 (1997)). ThenadX gene from Thermotoga maritime encoding for the aspartatedehydrogenase is involved in the biosynthesis of NAD (Yang et al. J.Biol. Chem. 278:8804-8808 (2003)).

Gene Accession No. GI No. Organism gdhA P00370 118547 Escherichia coligdh P96110.4 6226595 Thermotoga maritima gdhA1 NP_279651.1 15789827Halobacterium salinarum ldh P0A393 61222614 Bacillus cereus nadXNP_229443.1 15644391 Thermotoga maritima

The lysine 6-dehydrogenase (deaminating), encoded by lysDH gene,catalyze the oxidative deamination of the ε-amino group of L-lysine toform 2-aminoadipate-6-semialdehyde, which in turn nonenzymaticallycyclizes to form Δ1-piperideine-6-carboxylate (Misono and Nagasaki J.Bacteriol. 150:398-401 (1982)). The lysDH gene from Geobacillusstearothermophilus encodes a thermophilic NAD-dependent lysine6-dehydrogenase (Heydari et al. Appl Environ. Microbiol 70:937-942(2004)). In addition, the lysDH gene from Aeropyrum pernix K1 isidentified through homology from genome projects.

Gene Accession No. GI No. Organism lysDH BAB39707 13429872 Geobacillusstearothermophilus lysDH NP_147035.1 14602185 Aeropyrum pernix K1 ldhP0A393 61222614 Bacillus cereus2.3.1.a—Acyltransferase (Transferring Phosphate Group)

Exemplary phosphate transferring acyltransferases includephosphotransacetylase, encoded by pta, and phosphotransbutyrylase,encoded by ptb. The pta gene from E. coli encodes an enzyme that canconvert acetyl-CoA into acetyl-phosphate, and vice versa (Suzuki, T.Biochim. Biophys. Acta 191:559-569 (1969)). This enzyme can also utilizepropionyl-CoA instead of acetyl-CoA forming propionate in the process(Hesslinger et al. Mol. Microbiol 27:477-492 (1998)). Similarly, the ptbgene from C. acetobutylicum encodes an enzyme that can convertbutyryl-CoA into butyryl-phosphate (Walter et al. Gene 134(1): p. 107-11(1993)); Huang et al. J Mol Microbiol Biotechnol 2(1): p. 33-38 (2000).Additional ptb genes can be found in butyrate-producing bacterium L2-50(Louis et al. J. Bacteriol. 186:2099-2106 (2004)) and Bacillusmegaterium (Vazquez et al. Curr. Microbiol 42:345-349 (2001)).

Gene Accession No. GI No. Organism pta NP_416800.1 16130232 Escherichiacoli ptb NP_349676 15896327 Clostridium acetobutylicum ptb AAR19757.138425288 butyrate-producing bacterium L2-50 ptb CAC07932.1 10046659Bacillus megaterium2.6.1.a—Aminotransferase

Aspartate aminotransferase transfers an amino group from aspartate toalpha-ketoglutarate, forming glutamate and oxaloacetate. This conversionis catalyzed by, for example, the gene products of aspC from Escherichiacoli (Yagi et al. FEBS Lett. 100:81-84 (1979); Yagi et al. MethodsEnzymol. 113:83-89 (1985)), AAT2 from Saccharomyces cerevisiae (Yagi etal. J Biochem. 92:35-43 (1982)) and ASPS from Arabidopsis thaliana (48,108, 225 48. de la et al. Plant J 46:414-425 (2006); Kwok and Hanson JExp. Bot. 55:595-604 (2004); Wilkie and Warren Protein Expr. Purif.12:381-389 (1998)). Valine aminotransferase catalyzes the conversion ofvaline and pyruvate to 2-ketoisovalerate and alanine. The E. coli gene,avtA, encodes one such enzyme (Whalen and Berg J. Bacteriol. 150:739-746(1982)). This gene product also catalyzes the amination ofα-ketobutyrate to generate α-aminobutyrate, although the amine donor inthis reaction has not been identified (Whalen and Berg J. Bacteriol.158:571-574 (1984)). The gene product of the E. coli serC catalyzes tworeactions, phosphoserine aminotransferase and phosphohydroxythreonineaminotransferase (Lam and Winkler J. Bacteriol. 172:6518-6528 (1990)),and activity on non-phosphorylated substrates could not be detected(Drewke et al. FEBS. Lett. 390:179-182 (1996)).

Gene Accession No. GI No. Organism aspC NP_415448.1 16128895 Escherichiacoli AAT2 P23542.3 1703040 Saccharomyces cerevisiae ASP5 P46248.220532373 Arabidopsis thaliana avtA YP_026231.1 49176374 Escherichia coliserC NP_415427.1 16128874 Escherichia coli

Cargill has developed a beta-alanine/alpha-ketoglutarateaminotransferase for producing 3-HP from beta-alanine viamalonyl-semialdehyde (PCT/US2007/076252 (Jessen et al)). The geneproduct of SkPYD4 in Saccharomyces kluyveri was also shown topreferentially use beta-alanine as the amino group donor (Andersen etal. FEBS. J. 274:1804-1817 (2007)). SkUGA1 encodes a homologue ofSaccharomyces cerevisiae GABA aminotransferase, UGA1 (Ramos et al. Eur.J. Biochem. 149:401-404 (1985)), whereas SkPYD4 encodes an enzymeinvolved in both β-alanine and GABA transamination (Andersen et al.FEBS. J. 274:1804-1817 (2007)). 3-Amino-2-methylpropionate transaminasecatalyzes the transformation from methylmalonate semialdehyde to3-amino-2-methylpropionate. The enzyme has been characterized in Rattusnorvegicus and Sus scrofa and is encoded by Abat (Kakimoto et al.Biochim. Biophys. Acta 156:374-380 (1968); Tamaki et al. MethodsEnzymol. 324:376-389 (2000)). Enzyme candidates in other organisms withhigh sequence homology to 3-amino-2-methylpropionate transaminaseinclude Gta-1 in C. elegans and gabT in Bacillus subtilus. Additionally,one of the native GABA aminotransferases in E. coli, encoded by genegabT, has been shown to have broad substrate specificity (Liu et al.Biochemistry 43:10896-10905 (2004); Schulz et al. Appl Environ Microbiol56:1-6 (1990)). The gene product of puuE catalyzes the other4-aminobutyrate transaminase in E. coli (Kurihara et al. J. Biol. Chem.280:4602-4608 (2005)).

Gene Accession No. GI No. Organism SkyPYD4 ABF58893.1 98626772Saccharomyces kluyveri SkUGA1 ABF58894.1 98626792 Saccharomyces kluyveriUGA1 NP_011533.1 6321456 Saccharomyces cerevisiae Abat P50554.3122065191 Rattus norvegicus Abat P80147.2 120968 Sus scrofa Gta-1Q21217.1 6016091 Caenorhabditis elegans gabT P94427.1 6016090 Bacillussubtilus gabT P22256.1 120779 Escherichia coli K12 puuE NP_415818.116129263 Escherichia coli K12

The X-ray crystal structures of E. coli 4-aminobutyrate transaminaseunbound and bound to the inhibitor were reported (Liu et al.Biochemistry 43:10896-10905 (2004)). The substrates binding andsubstrate specificities were studied and suggested. The roles of activesite residues were studied by site-directed mutagenesis and X-raycrystallography (Liu et al. Biochemistry 44:2982-2992 (2005)). Based onthe structural information, attempt was made to engineer E. coli4-aminobutyrate transaminase with novel enzymatic activity. Thesestudies provide a base for evolving transaminase activity for BDOpathways.

2.7.2.a—Phosphotransferase, Carboxyl Group Acceptor

Exemplary kinases include the E. coli acetate kinase, encoded by ackA(Skarstedt and Silverstein J. Biol. Chem. 251:6775-6783 (1976)), the C.acetobutylicum butyrate kinases, encoded by buk1 and buk2 (Walter et al.Gene 134(1):107-111 (1993) (Huang et al. J Mol Microbiol Biotechnol2(1):33-38 (2000)], and the E. coli gamma-glutamyl kinase, encoded byproB (Smith et al. J. Bacteriol. 157:545-551 (1984)). These enzymesphosphorylate acetate, butyrate, and glutamate, respectively. The ackAgene product from E. coli also phosphorylates propionate (Hesslinger etal. Mol. Microbiol 27:477-492 (1998)).

Gene Accession No. GI No. Organism ackA NP_416799.1 16130231 Escherichiacoli buk1 NP_349675 15896326 Clostridium acetobutylicum buk2 Q97II120137415 Clostridium acetobutylicum proB NP_414777.1 16128228Escherichia coli2.8.3.a—Coenzyme-A Transferase

In the CoA-transferase family, E. coli enzyme acyl-CoA:acetate-CoAtransferase, also known as acetate-CoA transferase (EC 2.8.3.8), hasbeen shown to transfer the CoA moiety to acetate from a variety ofbranched and linear acyl-CoA substrates, including isobutyrate (Matthiesand Schink Appl Environ Microbiol 58:1435-1439 (1992)), valerate(Vanderwinkel et al. Biochem. Biophys. Res Commun. 33:902-908 (1968))and butanoate (Vanderwinkel, supra (1968)). This enzyme is encoded byatoA (alpha subunit) and atoD (beta subunit) in E. coli sp. K12 (Korolevet al. Acta Crystallogr. D Biol Crystallogr. 58:2116-2121 (2002);Vanderwinkel, supra (1968)) and actA and cg0592 in Corynebacteriumglutamicum ATCC 13032 (Duncan et al. Appl Environ Microbiol 68:5186-5190(2002)). Additional genes found by sequence homology include atoD andatoA in Escherichia coli UT189.

Gene Accession No. GI No. Organism atoA P76459.1 2492994 Escherichiacoli K12 atoD P76458.1 2492990 Escherichia coli K12 actA YP_226809.162391407 Corynebacterium glutamicum ATCC 13032 cg0592 YP_224801.162389399 Corynebacterium glutamicum ATCC 13032 atoA ABE07971.1 91073090Escherichia coli UT189 atoD ABE07970.1 91073089 Escherichia coli UT189

Similar transformations are catalyzed by the gene products of cat1,cat2, and cat3 of Clostridium kluyveri which have been shown to exhibitsuccinyl-CoA, 4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferaseactivity, respectively (Seedorf et al. Proc Natl Acad Sci U.S.A.105(6):2128-2133 (2008); Sohling and Gottschalk J Bacteriol178(3):871-880 (1996)].

Gene Accession No. GI No. Organism cat1 P38946.1 729048 Clostridiumkluyveri cat2 P38942.2 1705614 Clostridium kluyveri cat3 EDK35586.1146349050 Clostridium kluyveri

The glutaconate-CoA-transferase (EC 2.8.3.12) enzyme from anaerobicbacterium Acidaminococcus fermentans reacts with diacid glutaconyl-CoAand 3-butenoyl-CoA (Mack and Buckel FEBS Lett. 405:209-212 (1997)). Thegenes encoding this enzyme are gctA and gctB. This enzyme has reducedbut detectable activity with other CoA derivatives includingglutaryl-CoA, 2-hydroxyglutaryl-CoA, adipyl-CoA and acrylyl-CoA (Buckelet al. Eur. J. Biochem. 118:315-321 (1981)). The enzyme has been clonedand expressed in E. coli (Mac et al. Eur. J. Biochem. 226:41-51 (1994)).

Gene Accession No. GI No. Organism gctA CAA57199.1 559392Acidaminococcus fermentans gctB CAA57200.1 559393 Acidaminococcusfermentans3.1.2.a—Thiolester Hydrolase (CoA Specific)

In the CoA hydrolase family, the enzyme 3-hydroxyisobutyryl-CoAhydrolase is specific for 3-HIBCoA and has been described to efficientlycatalyze the desired transformation during valine degradation (Shimomuraet al. J Biol Chem 269:14248-14253 (1994)). Genes encoding this enzymeinclude hibch of Rattus norvegicus (Shimomura et al., supra (1994);Shimomura et al. Methods Enzymol. 324:229-240 (2000) and Homo sapiens(Shimomura et al., supra, 2000). Candidate genes by sequence homologyinclude hibch of Saccharomyces cerevisiae and BC_(—)2292 of Bacilluscereus.

Gene Accession No. GI No. Organism hibch Q5XIE6.2 146324906 Rattusnorvegicus hibch Q6NVY1.2 146324905 Homo sapiens hibch P28817.2 2506374Saccharomyces cerevisiae BC_2292 Q81DR3 81434808 Bacillus cereus

The conversion of adipyl-CoA to adipate can be carried out by anacyl-CoA hydrolase or equivalently a thioesterase. The top E. coli genecandidate is tesB (Naggert et al. J Biol Chem. 266(17):11044-11050(1991)] which shows high similarity to the human acot8 which is adicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westinet al. J Biol Chem 280(46): 38125-38132 (2005). This activity has alsobeen characterized in the rat liver (Deana, Biochem Int. 26(4): p.767-773 (1992)).

Gene Accession No. GI No. Organism tesB NP_414986 16128437 Escherichiacoli acot8 CAA15502 3191970 Homo sapiens acot8 NP_570112 51036669 Rattusnorvegicus

Other potential E. coli thioester hydrolases include the gene productsof tesA (Bonner and Bloch, J Biol Chem. 247(10):3123-3133 (1972)), ybgC(Kuznetsova et al., FEMS Microbiol Rev. 29(2):263-279 (2005); Zhuang etal., FEBS Lett. 516(1-3):161-163 (2002)) paaI (Song et al., J Biol Chem.281(16):11028-11038 (2006)), and ybdB (Leduc et al., J Bacteriol.189(19):7112-7126 (2007)).

Gene Accession No. GI No. Organism tesA NP_415027 16128478 Escherichiacoli ybgC NP_415264 16128711 Escherichia coli paaI NP_415914 16129357Escherichia coli ybdB NP_415129 16128580 Escherichia coli

Several eukaryotic acetyl-CoA hydrolases (EC 3.1.2.1) have broadsubstrate specificity. The enzyme from Rattus norvegicus brain (Robinsonet al. Biochem. Biophys. Res. Commun. 71:959-965 (1976)) can react withbutyryl-CoA, hexanoyl-CoA and malonyl-CoA.

Gene Accession No. GI No. Organism acot12 NP_570103.1 18543355 Rattusnorvegicus4.1.1.a—Carboxy-Lyase

An exemplary carboxy-lyase is acetolactate decarboxylase whichparticipates in citrate catabolism and branched-chain amino acidbiosynthesis, converting 2-acetolactate to acetoin. In Lactococcuslactis the enzyme is composed of six subunits, encoded by gene aldB, andis activated by valine, leucine and isoleucine (Goupil et al. Appl.Environ. Microbiol. 62:2636-2640 (1996); Goupil-Feuillerat et al. J.Bacteriol. 182:5399-5408 (2000)). This enzyme has been overexpressed andcharacterized in E. coli (Phalip et al. FEBS Lett. 351:95-99 (1994)). Inother organisms the enzyme is a dimer, encoded by aldC in Streptococcusthermophilus (Monnet et al. Lett. Appl. Microbiol. 36:399-405 (2003)),aldB in Bacillus brevis (Diderichsen et al. J. Bacteriol. 172:4315-4321(1990); Najmudin et al. Acta Crystallogr. D. Biol. Crystallogr.59:1073-1075 (2003)) and budA from Enterobacter aerogenes (Diderichsenet al. J. Bacteriol. 172:4315-4321 (1990)). The enzyme from Bacillusbrevis was cloned and overexpressed in Bacillus subtilis andcharacterized crystallographically (Najmudin et al. Acta Crystallogr. D.Biol. Crystallogr. 59:1073-1075 (2003)). Additionally, the enzyme fromLeuconostoc lactis has been purified and characterized but the gene hasnot been isolated (O'Sullivan et al. FEMS Microbiol. Lett. 194:245-249(2001)).

Gene Accession No. GI No. Organism aldB NP_267384.1 15673210 Lactococcuslactis aldC Q8L208 75401480 Streptococcus thermophilus aldB P23616.1113592 Bacillus brevis budA P05361.1 113593 Enterobacter aerogenes

Aconitate decarboxylase catalyzes the final step in itaconatebiosynthesis in a strain of Candida and also in the filamentous fungusAspergillus terreus (Bonnarme et al. J Bacteriol. 177:3573-3578 (1995);Willke and Vorlop Appl Microbiol Biotechnol 56:289-295 (2001)). Althoughitaconate is a compound of biotechnological interest, the aconitatedecarboxylase gene or protein sequence has not been reported to date.

4-oxalocronate decarboxylase has been isolated from numerous organismsand characterized. Genes encoding this enzyme include dmpH and dmpE inPseudomonas sp. (strain 600) (Shingler et al. J Bacteriol. 174:711-724(1992)), xylII and xylIII from Pseudomonas putida (Kato and Asano Arch.Microbiol 168:457-463 (1997); Lian and Whitman J. Am. Chem. Soc.116:10403-10411 (1994); Stanley et al. Biochemistry 39:3514 (2000)) andReut_B5691 and Reut_B5692 from Ralstonia eutropha JMP134 (Hughes et al.J Bacteriol. 158:79-83 (1984)). The genes encoding the enzyme fromPseudomonas sp. (strain 600) have been cloned and expressed in E. coli(Shingler et al. J Bacteriol. 174:711-724 (1992)).

Gene Accession No. GI No. Organism dmpH CAA43228.1 45685 Pseudomonas sp.CF600 dmpE CAA43225.1 45682 Pseudomonas sp. CF600 xylII YP_709328.1111116444 Pseudomonas putida xylIII YP_709353.1 111116469 Pseudomonasputida Reut_B5691 YP_299880.1 73539513 Ralstonia eutropha JMP134Reut_B5692 YP_299881.1 73539514 Ralstonia eutropha JMP134

An additional class of decarboxylases has been characterized thatcatalyze the conversion of cinnamate (phenylacrylate) and substitutedcinnamate derivatives to the corresponding styrene derivatives. Theseenzymes are common in a variety of organisms and specific genes encodingthese enzymes that have been cloned and expressed in E. coli are: pad 1from Saccharomyces cerevisae (Clausen et al. Gene 142:107-112 (1994)),pdc from Lactobacillus plantarum (Barthelmebs et al. Appl EnvironMicrobiol 67:1063-1069 (2001); Qi et al. Metab Eng 9:268-276 (2007);Rodriguez et al. J. Agric. Food Chem. 56:3068-3072 (2008)), pofK (pad)from Klebsiella oxytoca (Hashidoko et al. Biosci. Biotech. Biochem.58:217-218 (1994); Uchiyama et al. Biosci. Biotechnol. Biochem.72:116-123 (2008)), Pedicoccus pentosaceus (Barthelmebs et al. ApplEnviron Microbiol 67:1063-1069 (2001)), and padC from Bacillus subtilisand Bacillus pumilus (Lingen et al. Protein Eng 15:585-593 (2002)). Aferulic acid decarboxylase from Pseudomonas fluorescens also has beenpurified and characterized (Huang et al. J. Bacteriol. 176:5912-5918(1994)). Importantly, this class of enzymes have been shown to be stableand do not require either exogenous or internally bound co-factors, thusmaking these enzymes ideally suitable for biotransformations(Sariaslani, Annu. Rev. Microbiol. 61:51-69 (2007)).

Gene Accession No. GI No. Organism pad1 AB368798, BAG32372.1 188496948,188496949 Saccharomyces cerevisae pdc U63827, AAC45282.1 1762615,1762616 Lactobacillus plantarum pofK (pad) AB330293, BAF65031.1149941607, 149941608 Klebsiella oxytoca padC AF017117, AAC46254.12394281, 2394282 Bacillus subtilis pad AJ276891, CAC16794.1 11322456,11322458 Pedicoccus pentosaceus pad AJ278683, CAC18719.1 11691809,11691810 Bacillus pumilus

Additional decarboxylase enzymes can form succinic semialdehyde fromalpha-ketoglutarate. These include the alpha-ketoglutarate decarboxylaseenzymes from Euglena gracilis (Shigeoka et al. Biochem. J. 282(Pt2):319-323 (1992); Shigeoka and Nakano Arch. Biochem. Biophys. 288:22-28(1991); Shigeoka and Nakano Biochem. J. 292 (Pt 2):463-467 (1993)),whose corresponding gene sequence has yet to be determined, and fromMycobacterium tuberculosis (Tian et al. Proc Natl Acad Sci U.S.A.102:10670-10675 (2005)). In addition, glutamate decarboxylase enzymescan convert glutamate into 4-aminobutyrate such as the products of theE. coli gadA and gadB genes (De Biase et al. Protein. Expr. Purif.8:430-438 (1993)).

Gene Accession No. GI No. Organism kgd O50463.4 160395583 Mycobacteriumtuberculosis gadA NP_417974 16131389 Escherichia coli gadB NP_41601016129452 Escherichia coli

Keto-Acid Decarboxylases

Pyruvate decarboxylase (PDC, EC 4.1.1.1), also termed keto-aciddecarboxylase, is a key enzyme in alcoholic fermentation, catalyzing thedecarboxylation of pyruvate to acetaldehyde. This enzyme has a broadsubstrate range for aliphatic 2-keto acids including 2-ketobutyrate,2-ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (Berg et al.Science 318:1782-1786 (2007)). The PDC from Zymomonas mobilus, encodedby pdc, has been a subject of directed engineering studies that alteredthe affinity for different substrates (Siegert et al. Protein Eng DesSel 18:345-357 (2005)). The PDC from Saccharomyces cerevisiae has alsobeen extensively studied, engineered for altered activity, andfunctionally expressed in E. coli (Killenberg-Jabs et al. Eur. J.Biochem. 268:1698-1704 (2001); Li and Jordan Biochemistry 38:10004-10012(1999); ter Schure et al. Appl. Environ. Microbiol. 64:1303-1307(1998)). The crystal structure of this enzyme is available(Killenberg-Jabs Eur. J. Biochem. 268:1698-1704 (2001)). Otherwell-characterized PDC candidates include the enzymes from Acetobacterpasteurians (Chandra et al. Arch. Microbiol. 176:443-451 (2001)) andKluyveromyces lactis (Krieger et al. Eur. J. Biochem. 269:3256-3263(2002)).

Gene Accession No. GI No. Organism pdc P06672.1 118391 Zymomonas mobiluspdc1 P06169 30923172 Saccharomyces cerevisiae pdc Q8L388 75401616Acetobacter pasteurians pdc1 Q12629 52788279 Kluyveromyces lactis

Like PDC, benzoylformate decarboxylase (EC 4.1.1.7) has a broadsubstrate range and has been the target of enzyme engineering studies.The enzyme from Pseudomonas putida has been extensively studied andcrystal structures of this enzyme are available (Hasson et al.Biochemistry 37:9918-9930 (1998); Polovnikova et al. Biochemistry42:1820-1830 (2003)). Site-directed mutagenesis of two residues in theactive site of the Pseudomonas putida enzyme altered the affinity (Km)of naturally and non-naturally occurring substrates (Siegert Protein EngDes Sel 18:345-357 (2005)). The properties of this enzyme have beenfurther modified by directed engineering (Lingen et al. Protein Eng15:585-593 (2002)); Lingen Chembiochem 4:721-726 (2003)). The enzymefrom Pseudomonas aeruginosa, encoded by mdlC, has also beencharacterized experimentally (Barrowman et al. FEMS Microbiology Letters34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri,Pseudomonas fluorescens and other organisms can be inferred by sequencehomology or identified using a growth selection system developed inPseudomonas putida (Henning et al. Appl. Environ. Microbiol.72:7510-7517 (2006)).

Gene Accession No. GI No. Organism mdlC P20906.2 3915757 Pseudomonasputida mdlC Q9HUR2.1 81539678 Pseudomonas aeruginosa dpgB ABN80423.1126202187 Pseudomonas stutzeri ilvB-1 YP_260581.1 70730840 Pseudomonasfluorescens4.2.1.a—Hydro-Lyase

The 2-(hydroxymethyl)glutarate dehydratase of Eubacterium barkeri is anexemplary hydro-lyase. This enzyme has been studied in the context ofnicotinate catabolism and is encoded by hmd (Alhapel et al. Proc NatlAcad Sci USA 103:12341-12346 (2006)). Similar enzymes with high sequencehomology are found in Bacteroides capillosus, Anaerotruncus colihominis,and Natranaerobius thermophilius.

Gene Accession No. GI No. Organism hmd ABC88407.1 86278275 Eubacteriumbarkeri BACCAP_02294 ZP_02036683.1 154498305 Bacteroides capillosus ATCC29799 ANACOL_02527 ZP_02443222.1 167771169 Anaerotruncus colihominis DSM17241 NtherDRAFT_2368 ZP_02852366.1 169192667 Natranaerobiusthermophilus JW/NM-WN-LF

A second exemplary hydro-lyase is fumarate hydratase, an enzymecatalyzing the dehydration of malate to fumarate. A wealth of structuralinformation is available for this enzyme and researchers havesuccessfully engineered the enzyme to alter activity, inhibition andlocalization (Weaver, T. Acta Crystallogr. D Biol Crystallogr.61:1395-1401 (2005)). Additional fumarate hydratases include thoseencoded by fumC from Escherichia coli (Estevez et al. Protein Sci.11:1552-1557 (2002); Hong and Lee Biotechnol. Bioprocess Eng. 9:252-255(2004); Rose and Weaver Proc Natl Acad Sci US.A 101:3393-3397 (2004)),Campylobacter jejuni (Smith et al. Int. J Biochem. Cell Biol 31:961-975(1999)) and Thermus thermophilus (Mizobata et al. Arch. Biochem.Biophys. 355:49-55 (1998)), and fumH from Rattus norvegicus (Kobayashiet al. J Biochem. 89:1923-1931(1981)). Similar enzymes with highsequence homology include fum1 from Arabidopsis thaliana and fumC fromCorynebacterium glutamicum.

Gene Accession No. GI No. Organism fumC P05042.1 120601 Escherichia coliK12 fumC O69294.1 9789756 Campylobacter jejuni fumC P84127 75427690Thermus thermophilus fumH P14408.1 120605 Rattus norvegicus fum1P93033.2 39931311 Arabidopsis thaliana fumC Q8NRN8.1 39931596Corynebacterium glutamicum

Citramalate hydrolyase, also called 2-methylmalate dehydratase, converts2-methylmalate to mesaconate. 2-Methylmalate dehydratase activity wasdetected in Clostridium tetanomorphum, Morganella morganii, Citrobacteramalonaticus in the context of the glutamate degradation VI pathway(Kato and Asano Arch. Microbiol 168:457-463 (1997)); however the genesencoding this enzyme have not been sequenced to date.

The gene product of crt from C. acetobutylicum catalyzes the dehydrationof 3-hydroxybutyryl-CoA to crotonyl-CoA (Atsumi et al. Metab Eng.; 29(2007)); Boynton et al. Journal of Bacteriology 178:3015-3024 (1996)).The enoyl-CoA hydratases, phaA and phaB, of P. putida are believed tocarry out the hydroxylation of double bonds during phenylacetatecatabolism; (Olivera et al. Proc Natl Acad Sci USA 95(11):6419-6424(1998)). The paaA and paaB from P. fluorescens catalyze analogoustransformations (14 Olivera et al., supra, 1998). Lastly, a number ofEscherichia coli genes have been shown to demonstrate enoyl-CoAhydratase functionality including maoC (Park and Lee J Bacteriol185(18):5391-5397 (2003)), paaF (Park and Lee Biotechnol Bioeng.86(6):681-686 (2004a)); Park and Lee Appl Biochem Biotechnol. 113-116:335-346 (2004b)); Ismail et al. Eur J Biochem 270(14):p. 3047-3054(2003), and paaG (Park and Lee, supra, 2004; Park and Lee supra, 2004b;Ismail et al., supra, 2003).

Gene Accession No. GI No. Organism maoC NP_415905.1 16129348 Escherichiacoli paaF NP_415911.1 16129354 Escherichia coli paaG NP_415912.116129355 Escherichia coli crt NP_349318.1 15895969 Clostridiumacetobutylicum paaA NP_745427.1 26990002 Pseudomonas putida paaBNP_745426.1 26990001 Pseudomonas putida phaA ABF82233.1 106636093Pseudomonas fluorescens phaB ABF82234.1 106636094 Pseudomonasfluorescens

The E. coli genes fadA and fadB encode a multienzyme complex thatexhibits ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase, andenoyl-CoA hydratase activities (Yang et al. Biochemistry 30(27): p.6788-6795 (1991); Yang et al. J Biol Chem 265(18): p. 10424-10429(1990); Yang et al. J Biol Chem 266(24): p. 16255 (1991); Nakahigashiand Inokuchi Nucleic Acids Res 18(16): p. 4937 (1990)). The fadI andfadJ genes encode similar functions and are naturally expressed onlyanaerobically (Campbell et al. Mol Microbiol 47(3): p. 793-805 (2003). Amethod for producing poly[(R)-3-hydroxybutyrate] in E. coli thatinvolves activating fadB (by knocking out a negative regulator, fadR)and co-expressing a non-native ketothiolase (phaA from Ralstoniaeutropha) has been described previously (Sato et al. J Biosci Bioeng103(1): 38-44 (2007)). This work clearly demonstrates that a β-oxidationenzyme, in particular the gene product of fadB which encodes both3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities, canfunction as part of a pathway to produce longer chain molecules fromacetyl-CoA precursors.

Gene Accession No. GI No. Organism fadA YP_026272.1 49176430 Escherichiacoli fadB NP_418288.1 16131692 Escherichia coli fadI NP_416844.116130275 Escherichia coli fadJ NP_416843.1 16130274 Escherichia colifadR NP_415705.1 16129150 Escherichia coli4.3.1.a—Ammonia-Lyase

Aspartase (EC 4.3.1.1), catalyzing the deamination of aspartate tofumarate, is a widespread enzyme in microorganisms, and has beencharacterized extensively (Viola, R. E. Adv. Enzymol. Relat Areas Mol.Biol 74:295-341 (2000)). The crystal structure of the E. coli aspartase,encoded by aspA, has been solved (Shi et al. Biochemistry 36:9136-9144(1997)). The E. coli enzyme has also been shown to react with alternatesubstrates aspartatephenylmethylester, asparagine, benzyl-aspartate andmalate (Ma et al. Ann N.Y. Acad Sci 672:60-65 (1992)). In a separatestudy, directed evolution was been employed on this enzyme to altersubstrate specificity (Asano et al. Biomol. Eng 22:95-101 (2005)).Enzymes with aspartase functionality have also been characterized inHaemophilus influenzae (Sjostrom et al. Biochim. Biophys. Acta1324:182-190 (1997)), Pseudomonas fluorescens (Takagi et al. J. Biochem.96:545-552 (1984)), Bacillus subtilus (Sjostrom et al. Biochim. Biophys.Acta 1324:182-190 (1997)) and Serratia marcescens (Takagi and Kisumi JBacteriol. 161:1-6 (1985)).

Gene Accession No. GI No. Organism aspA NP_418562 90111690 Escherichiacoli K12 subsp. MG1655 aspA P44324.1 1168534 Haemophilus influenzae aspAP07346.1 114273 Pseudomonas fluorescens ansB P26899.1 114271 Bacillussubtilus aspA P33109.1 416661 Serratia marcescens

3-methylaspartase (EC 4.3.1.2), also known as beta-methylaspartase or3-methylaspartate ammonia-lyase, catalyzes the deamination ofthreo-3-methylaspartate to mesaconate. The 3-methylaspartase fromClostridium tetanomorphum has been cloned, functionally expressed in E.coli, and crystallized (Asuncion et al. Acta Crystallogr. D BiolCrystallogr. 57:731-733 (2001); Asuncion et al. J Biol Chem.277:8306-8311 (2002); Botting et al. Biochemistry 27:2953-2955 (1988);Goda et al. Biochemistry 31:10747-10756 (1992). In Citrobacteramalonaticus, this enzyme is encoded by BAA28709 (Kato and Asano Arch.Microbiol 168:457-463 (1997)). 3-Methylaspartase has also beencrystallized from E. coli YG1002 (Asano and Kato FEMS Microbiol Lett.118:255-258 (1994)) although the protein sequence is not listed inpublic databases such as GenBank. Sequence homology can be used toidentify additional candidate genes, including CTC_(—)02563 in C. tetaniand ECs0761 in Escherichia coli O157:H7.

Gene Accession No. GI No. Organism MAL AAB24070.1 259429 Clostridiumtetanomorphum BAA28709 BAA28709.1 3184397 Citrobacter amalonaticusCTC_02563 NP_783085.1 28212141 Clostridium tetani ECs0761 BAB34184.113360220 Escherichia coli O157:H7 str. Sakai

Ammonia-lyase enzyme candidates that form enoyl-CoA products includebeta-alanyl-CoA ammonia-lyase (EC 4.3.1.6), which deaminatesbeta-alanyl-CoA, and 3-aminobutyryl-CoA ammonia-lyase (EC 4.3.1.14). Twobeta-alanyl-CoA ammonia lyases have been identified and characterized inClostridium propionicum (Herrmann et al. FEBS J. 272:813-821 (2005)). Noother beta-alanyl-CoA ammonia lyases have been studied to date, but genecandidates can be identified by sequence similarity. One such candidateis MXAN_(—)4385 in Myxococcus xanthus.

Gene Accession No. GI No. Organism ac12 CAG29275.1 47496504 Clostridiumpropionicum acl1 CAG29274.1 47496502 Clostridium propionicum MXAN_4385YP_632558.1 108756898 Myxococcus xanthus5.3.3.a—Isomerase

The 4-hydroxybutyryl-CoA dehydratases from both Clostridiumaminobutyrium and C. kluyveri catalyze the reversible conversion of4-hydroxybutyryl-CoA to crotonyl-CoA and posses an intrinsicvinylacetyl-CoA Δ-isomerase activity (Scherf and Buckel Eur. J Biochem.215:421-429 (1993); Scherf et al. Arch. Microbiol 161:239-245 (1994)).Both native enzymes were purified and characterized, including theN-terminal amino acid sequences (Scherf and Buckel, supra, 1993; Scherfet al., supra, 1994). The abfD genes from C. aminobutyrium and C.kluyveri match exactly with these N-terminal amino acid sequences, thusare encoding the 4-hydroxybutyryl-CoA dehydratases/vinylacetyl-CoAΔ-isomerase. In addition, the abfD gene from Porphyromonas gingivalisATCC 33277 is identified through homology from genome projects.

Gene Accession No. GI No. Organism abfD YP_001396399.1 153955634Clostridium kluyveri DSM 555 abfD P55792 84028213 Clostridiumaminobutyricum abfD YP_001928843 188994591 Porphyromonas gingivalis ATCC332775.4.3.a—Aminomutase

Lysine 2,3-aminomutase (EC 5.4.3.2) is an exemplary aminomutase thatconverts lysine to (3S)-3,6-diaminohexanoate, shifting an amine groupfrom the 2- to the 3-position. The enzyme is found in bacteria thatferment lysine to acetate and butyrate, including as Fusobacteriumnuleatum (kamA) (Barker et al. J. Bacteriol. 152:201-207 (1982)) andClostridium subterminale (kamA) (Chirpich et al. J. Biol. Chem.245:1778-1789 (1970)). The enzyme from Clostridium subterminale has beencrystallized (Lepore et al. Proc. Natl. Acad. Sci. U.S.A 102:13819-13824(2005)). An enzyme encoding this function is also encoded by yodO inBacillus subtilus (Chen et al. Biochem. J. 348 Pt 3:539-549 (2000)). Theenzyme utilizes pyridoxal 5′-phosphate as a cofactor, requiresactivation by S-Adenosylmethoionine, and is stereoselective, reactingwith the only with L-lysine. The enzyme has not been shown to react withalternate substrates.

Gene Accession No. GI No. Organism yodO O34676.1 4033499 Bacillussubtilus kamA Q9XBQ8.1 75423266 Clostridium subterminale kamA Q8RHX481485301 Fusobacterium nuleatum subsp. nuleatum

A second aminomutase, beta-lysine 5,6-aminomutase (EC 5.4.3.3),catalyzes the next step of lysine fermentation to acetate and butyrate,which transforms (3S)-3,6-diaminohexanoate to(3S,5S)-3,5-diaminohexanoate, shifting a terminal amine group from the6- to the 5-position. This enzyme also catalyzes the conversion oflysine to 2,5-diaminohexanoate and is also called lysine-5,6-aminomutase(EC 5.4.3.4). The enzyme has been crystallized in Clostridiumsticklandii (kamD, kamE) (Berkovitch et al. Proc. Natl. Acad. Sci. U.S.A101:15870-15875 (2004)). The enzyme from Porphyromonas gingivalis hasalso been characterized (Tang et al. Biochemistry 41:8767-8776 (2002)).

Gene Accession No. GI No. Organism kamD AAC79717.1  3928904 Clostridiumsticklandii kamE AAC79718.1  3928905 Clostridium sticklandii kamDNC_002950.2, 34539880, Porphyromonas gingivalis W83 NP_905288.1 34540809kamE NC_002950.2, 34539880, Porphyromonas gingivalis W83 NP_905289.134540810

Ornithine 4,5-aminomutase (EC 5.4.3.5) converts D-ornithine to2,4-diaminopentanoate, also shifting a terminal amine to the adjacentcarbon. The enzyme from Clostridium sticklandii is encoded by two genes,oraE and oraS, and has been cloned, sequenced and expressed in E. coli(Chen et al. J. Biol. Chem. 276:44744-44750 (2001)). This enzyme has notbeen characterized in other organisms to date.

Gene Accession No. GI No. Organism oraE AAK72502 17223685 Clostridiumsticklandii oraS AAK72501 17223684 Clostridium sticklandii

Tyrosine 2,3-aminomutase (EC 5.4.3.6) participates in tyrosinebiosynthesis, reversibly converting tyrosine to3-amino-3-(4-hydroxyphenyl)propanoate by shifting an amine from the 2-to the 3-position. In Streptomyces globisporus the enzyme has also beenshown to react with tyrosine derivatives (Christenson et al.Biochemistry 42:12708-12718 (2003)). Sequence information is notavailable.

Leucine 2,3-aminomutase (EC 5.4.3.7) converts L-leucine to beta-leucineduring leucine degradation and biosynthesis. An assay for leucine2,3-aminomutase detected activity in many organisms (Poston, J. M.Methods Enzymol. 166:130-135 (1988)) but genes encoding the enzyme havenot been identified to date.

Cargill has developed a novel 2,3-aminomutase enzyme to convertL-alanine to β-alanine, thus creating a pathway from pyruvate to 3-HP infour biochemical steps (Liao et al., U.S. Publication No. 2005-0221466).

6.2.1.a—Acid-Thiol Ligase

An exemplary acid-thiol ligase is the gene products of sucCD of E. coliwhich together catalyze the formation of succinyl-CoA from succinatewith the concaminant consumption of one ATP, a reaction which isreversible in vivo (Buck et al. Biochemistry 24(22): p. 6245-6252(1985)). Additional exemplary CoA-ligases include the ratdicarboxylate-CoA ligase for which the sequence is yet uncharacterized(Vamecq et al. Biochem J. 230(3): p. 683-693 (1985)), either of the twocharacterized phenylacetate-CoA ligases from P. chrysogenum(Lamas-Maceiras et al. Biochem J395(1):147-155 (2006); Wang et al.Biochem Biophys Res Commun, 360(2):453-458 (2007)), thephenylacetate-CoA ligase from Pseudomonas putida (Martinez-Blanco et al.J Biol Chem. 265(12):7084-7090 (1990)), and the 6-carboxyhexanoate-CoAligase from Bacillus subtilis (Bower et al. J Bacteriol178(14):4122-4130 (1996)).

Gene Accession No. GI No. Organism sucC NP_415256.1 16128703 Escherichiacoli sucD AAC73823.1 1786949 Escherichia coli phl CAJ15517.1 77019264Penicillium chrysogenum phlB ABS19624.1 152002983 Penicilliumchrysogenum paaF AAC24333.2 22711873 Pseudomonas putida bioW NP_390902.250812281 Bacillus subtilis

Example V Exemplary BDO Pathway from Succinyl-CoA

This example describes exemplary BDO pathways from succinyl-CoA.

BDO pathways from succinyl-CoA are described herein and have beendescribed previously (see U.S. application Ser. No. 12/049,256, filedMar. 14, 2008, and PCT application Ser. No. 08/57,168, filed Mar. 14,2008, each of which is incorporated herein by reference). Additionalpathways are shown in FIG. 8A. Enzymes of such exemplary BDO pathwaysare listed in Table 15, along with exemplary genes encoding theseenzymes.

Briefly, succinyl-CoA can be converted to succinic semialdehyde bysuccinyl-CoA reductase (or succinate semialdehyde dehydrogenase) (EC1.2.1.b). Succinate semialdehyde can be converted to 4-hydroxybutyrateby 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a), as previouslydescribed. Alternatively, succinyl-CoA can be converted to4-hydroxybutyrate by succinyl-CoA reductase (alcohol forming) (EC1.1.1.c). 4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-CoA by4-hydroxybutyryl-CoA transferase (EC 2.8.3.a), as previously described,or by 4-hydroxybutyryl-CoA hydrolase (EC 3.1.2.a) or4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoA synthetase) (EC6.2.1.a). Alternatively, 4-hydroxybutyrate can be converted to4-hydroxybutyryl-phosphate by 4-hydroxybutyrate kinase (EC 2.7.2.a), aspreviously described. 4-Hydroxybutyryl-phosphate can be converted to4-hydroxybutyryl-CoA by phosphotrans-4-hydroxybutyrylase (EC 2.3.1.a),as previously described. Alternatively, 4-hydroxybutyryl-phosphate canbe converted to 4-hydroxybutanal by 4-hydroxybutanal dehydrogenase(phosphorylating) (EC 1.2.1.d) (acylphosphate reductase).4-Hydroxybutyryl-CoA can be converted to 4-hydroxybutanal by4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase) (EC1.2.1.b). Alternatively, 4-hydroxybutyryl-CoA can be converted to1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol forming) (EC1.1.1.c). 4-Hydroxybutanal can be converted to 1,4-butanediol by1,4-butanediol dehydrogenase (EC 1.1.1.a), as previously described.

TABLE 15 BDO pathway from succinyl-CoA. EC Desired GenBank ID Figureclass substrate Desired product Enzyme name Gene name (if available)Organism Known Substrates 8A 1.2.1.b succinyl-CoA succinic succinyl-CoAsucD P38947.1 Clostridium succinyl-CoA semialdehyde reductase kluyveri(or succinate semialdehyde dehydrogenase) sucD NP_904963.1 Porphyromonassuccinyl-CoA gingivalis Msed_0709 YP_001190808.1 Metallosphaeramalonyl-CoA sedula 8A 1.1.1.a succinate 4- 4-hydroxybutyrate 4hbdYP_726053.1 Ralstonia 4-hydroxybutyrate semialdehyde hydroxybutyratedehydrogenase eutropha H16 4hbd L21902.1 Clostridium 4-hydroxybutyratekluyveri DSM 555 4hbd Q94B07 Arabidopsis 4-hydroxybutyrate thaliana 8A1.1.1.c succinyl-CoA 4- succinyl-CoA adhE2 AAK09379.1 Clostridiumbutanoyl-CoA hydroxybutyrate reductase acetobutylicum (alcohol forming)mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1Simmondsia long chain acyl- chinensis CoA 8A 2.8.3.a 4- 4-4-hydroxybutyryl- cat1, cat2, P38946.1, Clostridium succinate, 4-hydroxybutyrate hydroxybutyryl- CoA transferase cat3 P38942.2, kluyverihydroxybutyrate, CoA EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia coli butanoate P76458.1 8A 3.1.2.a 4- 4- 4-hydroxybutyryl-tesB NP_414986 Escherichia coli adipyl-CoA hydroxybutyratehydroxybutyryl- CoA hydrolase CoA acot12 NP_570103.1 Rattus butyryl-CoAnorvegicus hibch Q6NVY1.2 Homo sapiens 3- hydroxypropanoyl- CoA 8A6.2.1.a 4- 4- 4-hydroxybutyryl- sucCD NP_415256.1, Escherichia colisuccinate hydroxybutyrate hydroxybutyryl- CoA ligase (or AAC73823.1 CoA4-hydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicilliumphenylacetate chrysogenum bioW NP_390902.2 Bacillus subtilis 6-carboxyhexanoate 8A 2.7.2.a 4- 4- 4-hydroxybutyryl- ackA NP_416799.1Escherichia coli acetate, propionate hydroxybutyrate hydroxybutyryl-kinase phosphate buk1 NP_349675 Clostridium butyrate acetobutylicum buk2Q97II1 Clostridium butyrate acetobutylicum 8A 2.3.1.a 4- 4-phosphotrans-4- ptb NP_349676 Clostridium butyryl-phosphatehydroxybutyryl- hydroxybutyryl- hydroxybutyrylase acetobutylicumphosphate CoA ptb AAR19757.1 butyrate- butyryl-phosphate producingbacterium L2-50 ptb CAC07932.1 Bacillus butyryl-phosphate megaterium 8A1.2.1.d 4- 4- 4-hydroxybutanal asd NP_417891.1 Escherichia coliL-4-aspartyl- hydroxybutyryl- hydroxybutanal dehydrogenase phosphatephosphate (phosphorylating) proA NP_414778.1 Escherichia coliL-glutamyl-5- phospate gapA P0A9B2.2 Escherichia coli Glyceraldehyde-3-phosphate 8A 1.2.1.b 4- 4- 4-hydroxybutyryl- sucD P38947.1 Clostridiumsuccinyl-CoA hydroxybutyryl- hydroxybutanal CoA reductase kluyveri CoA(or 4- hydroxybutanal dehydrogenase) sucD NP_904963.1 Porphyromonassuccinyl-CoA gingivalis Msed_0709 YP_001190808.1 Metallosphaeramalonyl-CoA sedula 8A 1.1.1.c 4- 1,4-butanediol 4-hydroxybutyryl- adhE2AAK09379.1 Clostridium butanoyl-CoA hydroxybutyryl- CoA reductaseacetobutylicum CoA (alcohol forming) mcr AAS20429.1 Chloroflexusmalonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long chain acyl-chinensis CoA 8A 1.1.1.a 4- 1,4-butanediol 1,4-butanediol ADH2NP_014032.1 Saccharymyces general hydroxybutanal dehydrogenasecerevisiae yqhD NP_417484.1 Escherichia coli >C3 4hbd L21902.1Clostridium Succinate kluyveri semialdehyde DSM 555

Example VI Additional Exemplary BDO Pathways from Alpha-Ketoglutarate

This example describes exemplary BDO pathways from alpha-ketoglutarate.

BDO pathways from succinyl-CoA are described herein and have beendescribed previously (see U.S. application Ser. No. 12/049,256, filedMar. 14, 2008, and PCT application Ser. No. 08/57,168, filed Mar. 14,2008, each of which is incorporated herein by reference). Additionalpathways are shown in FIG. 8B. Enzymes of such exemplary BDO pathwaysare listed in Table 16, along with exemplary genes encoding theseenzymes.

Briefly, alpha-ketoglutarate can be converted to succinic semialdehydeby alpha-ketoglutarate decarboxylase (EC 4.1.1.a), as previouslydescribed. Alternatively, alpha-ketoglutarate can be converted toglutamate by glutamate dehydrogenase (EC 1.4.1.a). 4-Aminobutyrate canbe converted to succinic semialdehyde by 4-aminobutyrate oxidoreductase(deaminating) (EC 1.4.1.a) or 4-aminobutyrate transaminase (EC 2.6.1.a).Glutamate can be converted to 4-aminobutyrate by glutamate decarboxylase(EC 4.1.1.a). Succinate semialdehyde can be converted to4-hydroxybutyrate by 4-hydroxybutyrate dehydrogenase (EC 1.1.1.a), aspreviously described. 4-Hydroxybutyrate can be converted to4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA transferase (EC 2.8.3.a),as previously described, or by 4-hydroxybutyryl-CoA hydrolase (EC3.1.2.a), or 4-hydroxybutyryl-CoA ligase (or 4-hydroxybutyryl-CoAsynthetase) (EC 6.2.1.a). 4-Hydroxybutyrate can be converted to4-hydroxybutyryl-phosphate by 4-hydroxybutyrate kinase (EC 2.7.2.a).4-Hydroxybutyryl-phosphate can be converted to 4-hydroxybutyryl-CoA byphosphotrans-4-hydroxybutyrylase (EC 2.3.1.a), as previously described.Alternatively, 4-hydroxybutyryl-phosphate can be converted to4-hydroxybutanal by 4-hydroxybutanal dehydrogenase (phosphorylating) (EC1.2.1.d) (acylphosphate reductase). 4-Hydroxybutyryl-CoA can beconverted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or4-hydroxybutanal dehydrogenase) (EC 1.2.1.b), as previously described.4-Hydroxybutyryl-CoA can be converted to 1,4-butanediol by4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).4-Hydroxybutanal can be converted to 1,4-butanediol by 1,4-butanedioldehydrogenase (EC 1.1.1.a), as previously described.

TABLE 16 BDO pathway from alpha-ketoglutarate. Fig- EC Desired GenBankID Known ure class substrate Desired product Enzyme name Gene name (ifavailable) Organism Substrates 8B 4.1.1.a alpha- succinic alpha- kgdO50463.4 Mycobacterium alpha- ketoglutarate semialdehyde ketoglutaratetuberculosis ketoglutarate decarboxylase gadA NP_417974 Escherichiaglutamate coli gadB NP_416010 Escherichia glutamate coli 8B 1.4.1.aalpha- glutamate glutamate gdhA P00370 Escherichia glutamateketoglutarate dehydrogenase coli gdh P96110.4 Thermotoga glutamatemaritima gdhA1 NP_279651.1 Halobacterium glutamate salinarum 8B 1.4.1.a4- succinic 4-aminobutyrate lysDH AB052732 Geobacillus lysineaminobutyrate semialdehyde oxidoreductase stearothermophilus(deaminating) lysDH NP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393Bacillus cereus leucine, isoleucine, valine, 2- aminobutanoate 8B2.6.1.a 4- succinic 4-aminobutyrate gabT P22256.1 Escherichia 4-aminobutyrate semialdehyde transaminase coli aminobutyryate puuENP_415818.1 Escherichia 4- coli aminobutyryate UGA1 NP_011533.1Saccharomyces 4- cerevisiae aminobutyryate 8B 4.1.1.a glutamate 4-glutamate gadA NP_417974 Escherichia glutamate aminobutyratedecarboxylase coli gadB NP_416010 Escherichia glutamate coli kgdO50463.4 Mycobacterium alpha- tuberculosis ketoglutarate 8B 1.1.1.asuccinate 4- 4-hydroxybutyrate 4hbd YP_726053.1 Ralstonia 4-semialdehyde hydroxybutyrate dehydrogenase eutropha H16 hydroxybutyrate4hbd L21902.1 Clostridium 4- kluyveri hydroxybutyrate DSM 555 4hbdQ94B07 Arabidopsis 4- thaliana hydroxybutyrate 8B 2.8.3.a 4- 4-4-hydroxybutyryl- cat1, cat2, P38946.1, Clostridium succinate, 4-hydroxybutyrate hydroxybutyryl- CoA transferase cat3 P38942.2, kluyverihydroxybutyrate, CoA EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia butanoate P76458.1 coli 8B 3.1.2.a 4- 4- 4-hydroxybutyryl-tesB NP_414986 Escherichia adipyl-CoA hydroxybutyrate hydroxybutyryl-CoA hydrolase coli CoA acot12 NP_570103.1 Rattus butyryl-CoA norvegicushibch Q6NVYL.2 Homo sapiens 3- hydroxypropanoyl- CoA 8B 6.2.1.a 4- 4-4-hydroxybutyryl- sucCD NP_415256.1, Escherichia succinatehydroxybutyrate hydroxybutyryl- CoA ligase (or 4- AAC73823.1 coli CoAhydroxybutyryl- CoA synthetase) phl CAJ15517.1 Penicillium phenylacetatechrysogenum bioW NP_390902.2 Bacillus subtilis 6- carboxyhexanoate 8B2.7.2.a 4- 4- 4-hydroxybutyrate ackA NP_416799.1 Escherichia acetate,hydroxybutyrate hydroxybutyryl- kinase coli propionate phosphate buk1NP_349675 Clostridium butyrate acetobutylicum buk2 Q97II1 Clostridiumbutyrate acetobutylicum 8B 2.3.1.a 4- 4- phosphotrans-4- ptb NP_349676Clostridium butyryl- hydroxybutyryl- hydroxybutyryl- hydroxybutyrylaseacetobutylicum phosphate phosphate CoA ptb AAR19757.1 butyrate- butyryl-producing phosphate bacterium L2-50 ptb CAC07932.1 Bacillus butyryl-megaterium phosphate 8B 1.2.1.d 4- 4- 4-hydroxybutanal asd NP_417891.1Escherichia L-4-aspartyl- hydroxybutyryl- hydroxybutanal dehydrogenasecoli phosphate phosphate (phosphorylating) proA NP_414778.1 EscherichiaL-glutamyl-5- coli phospate gapA P0A9B2.2 Escherichia Glyceraldehyde-coli 3-phosphate 8B 1.2.1.b 4- 4- 4-hydroxybutyryl- sucD P38947.1Clostridium succinyl-CoA hydroxybutyryl- hydroxybutanal CoA reductase(or kluyveri CoA 4-hydroxybutanal dehydrogenase) sucD NP_904963.1Porphyromonas succinyl-CoA gingivalis Msed_0709 YP_001190808.1Metallosphaera malonyl-CoA sedula 8B 1.1.1.c 4- 1,4-butanediol4-hydroxybutyryl- adhE2 AAK09379.1 Clostridium butanoyl-CoAhydroxybutyryl- CoA reductase acetobutylicum CoA (alcohol forming) mcrAAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1Simmondsia long chain acyl- chinensis CoA 8B 1.1.1.a 4- 1,4-butanediol1,4-butanediol ADH2 NP_014032.1 Saccharymyces general hydroxybutanaldehydrogenase cerevisiae yqhD NP_417484.1 Escherichia >C3 coli 4hbdL21902.1 Clostridium Succinate kluyveri semialdehyde DSM 555

Example VII BDO Pathways from 4-Aminobutyrate

This example describes exemplary BDO pathways from 4-aminobutyrate.

FIG. 9A depicts exemplary BDO pathways in which 4-aminobutyrate isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 17, along with exemplary genes encoding these enzymes.

Briefly, 4-aminobutyrate can be converted to 4-aminobutyryl-CoA by4-aminobutyrate CoA transferase (EC 2.8.3.a), 4-aminobutyryl-CoAhydrolase (EC 3.1.2.a), or 4-aminobutyrate-CoA ligase (or4-aminobutyryl-CoA synthetase) (EC 6.2.1.a). 4-aminobutyryl-CoA can beconverted to 4-oxobutyryl-CoA by 4-aminobutyryl-CoA oxidoreductase(deaminating) (EC 1.4.1.a) or 4-aminobutyryl-CoA transaminase (EC2.6.1.a). 4-oxobutyryl-CoA can be converted to 4-hydroxybutyryl-CoA by4-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.a). 4-hydroxybutyryl-CoAcan be converted to 1,4-butanediol by 4-hydroxybutyryl-CoA reductase(alcohol forming) (EC 1.1.1.c). Alternatively, 4-hydroxybutyryl-CoA canbe converted to 4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or4-hydroxybutanal dehydrogenase) (EC 1.2.1.b). 4-hydroxybutanal can beconverted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC1.1.1.a).

TABLE 17 BDO pathway from 4-aminobutyrate. Desired Desired GenBank IDKnown Figure EC class substrate product Enzyme name Gene name (ifavailable) Organism Substrates 9A 2.8.3.a 4- 4- 4-aminobutyrate cat1,cat2, P38946.1, Clostridium succinate, 4- amino- aminobutyryl- CoAtransferase cat3 P38942.2, kluyveri hydroxybutyrate, butyrate CoAEDK35586.1 butyrate gctA, gctB CAA57199.1, Acidaminococcus glutarateCAA57200.1 fermentans atoA, atoD P76459.1, Escherichia coli butanoateP76458.1 9A 3.1.2.a 4- 4- 4-aminobutyryl- tesB NP_414986 Escherichiacoli adipyl-CoA amino- aminobutyryl- CoA hydrolase butyrate CoA acot12NP_570103.1 Rattus norvegicus butyryl-CoA hibch Q6NVY1.2 Homo sapiens 3-hydroxypro- panoyl-CoA 9A 6.2.1.a 4- 4- 4-aminobutyrate- sucCDNP_415256.1, Escherichia coli succinate amino- aminobutyryl- CoA ligase(or 4- AAC73823.1 butyrate CoA aminobutyryl- CoA synthetase) phlCAJ15517.1 Penicillium phenylacetate chrysogenum bioW NP_390902.2Bacillus subtilis 6- carboxyhexanoate 9A 1.4.1.a 4- 4-oxobutyryl-4-aminobutyryl- lysDH AB052732 Geobacillus lysine amino- CoA CoAstearothermophilus butyryl- oxidoreductase CoA (deaminating) lysDHNP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393 Bacillus cereusleucine, isoleucine, valine, 2- aminobutanoate 9A 2.6.1.a 4-4-oxobutyryl- 4-aminobutyryl- gabT P22256.1 Escherichia coli4-aminobutyryate amino- CoA CoA butyryl- transaminase CoA abat P50554.3Rattus norvegicus 3-amino-2- methylpropionate SkyPYD4 ABF58893.1Saccharomyces beta-alanine kluyveri 9A 1.1.1.a 4-oxo- 4-4-hydroxybutyryl- ADH2 NP_014032.1 Saccharymyces general butyryl-hydroxybutyryl- CoA cerevisiae CoA CoA dehydrogenase yqhD NP_417484.1Escherichia coli >C3 4hbd L21902.1 Clostridium kluyveri Succinate DSM555 semialdehyde 8 1.1.1.c 4- 1,4-butanediol 4-hydroxybutyryl- adhE2AAK09379.1 Clostridium butanoyl-CoA hydroxy- CoA reductaseacetobutylicum butyryl- (alcohol forming) CoA mcr AAS20429.1Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia longchain acyl- chinensis CoA 8 1.2.1.b 4- 4- 4-hydroxybutyryl- sucDP38947.1 Clostridium Succinyl-CoA hydroxy- hydroxybutanal CoA reductase(or kluyveri butyryl- 4-hydroxybutanal CoA dehydrogenase) sucDNP_904963.1 Porphyromonas Succinyl-CoA gingivalis Msed_ YP_Metallosphaera Malonyl- 0709 001190808.1 sedula CoA 8 1.1.1.a 4-1,4-butanediol 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalhydroxy- dehydrogenase cerevisiae butanal yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium kluyveri Succinate DSM 555semialdehyde

Enzymes for another exemplary BDO pathway converting 4-aminobutyrate toBDO is shown in FIG. 9A. Enzymes of such an exemplary BDO pathway arelisted in Table 18, along with exemplary genes encoding these enzymes.

Briefly, 4-aminobutyrate can be converted to 4-aminobutyryl-CoA by4-aminobutyrate CoA transferase (EC 2.8.3.a), 4-aminobutyryl-CoAhydrolase (EC 3.1.2.a) or 4-aminobutyrate-CoA ligase (or4-aminobutyryl-CoA synthetase) (EC 6.2.1.a). 4-aminobutyryl-CoA can beconverted to 4-aminobutan-1-ol by 4-aminobutyryl-CoA reductase (alcoholforming) (EC 1.1.1.c). Alternatively, 4-aminobutyryl-CoA can beconverted to 4-aminobutanal by 4-aminobutyryl-CoA reductase (or4-aminobutanal dehydrogenase) (EC 1.2.1.b), and 4-aminobutanal convertedto 4-aminobutan-1-ol by 4-aminobutan-1-ol dehydrogenase (EC 1.1.1.a).4-aminobutan-1-ol can be converted to 4-hydroxybutanal by4-aminobutan-1-ol oxidoreductase (deaminating) (EC 1.4.1.a) or4-aminobutan-1-ol transaminase (EC 2.6.1.a). 4-hydroxybutanal can beconverted to 1,4-butanediol by 1,4-butanediol dehydrogenase (EC1.1.1.a).

TABLE 18 BDO pathway from 4-aminobutyrate. Desired Desired GenBank IDKnown Figure EC class substrate product Enzyme name Gene name (ifavailable) Organism Substrate 9A 2.8.3.a 4- 4- 4-aminobutyrate cat1,cat2, P38946.1, Clostridium succinate, 4- amino- aminobutyryl- CoA cat3P38942.2, kluyveri hydroxybutyrate, butyrate CoA transferase EDK35586.1butyrate gctA, gctB CAA57199.1, Acidaminococcus glutarate CAA57200.1fermentans atoA, atoD P76459.1, Escherichia coli butanoate P76458.1 9A3.1.2.a 4- 4- 4-aminobutyryl- tesB NP_414986 Escherichia coli adipyl-CoAamino- aminobutyryl- CoA butyrate CoA hydrolase acot12 NP_570103.1Rattus norvegicus butyryl-CoA hibch Q6NVY1.2 Homo sapiens 3-hydroxypropanoyl- CoA 9A 6.2.1.a 4- 4- 4-aminobutyrate- sucCDNP_415256.1, Escherichia coli succinate amino- aminobutyryl- CoAAAC73823.1 butyrate CoA ligase (or 4- aminobutyryl- CoA synthetase) phlCAJ15517.1 Penicillium phenylacetate chrysogenum bioW NP_390902.2Bacillus subtilis 6- carboxyhexanoate 9A 1.1.1.c 4- 4-aminobutan-4-aminobutyryl- adhE2 AAK09379.1 Clostridium butanoyl-CoA amino- 1-olCoA acetobutylicum butyryl- reductase (alcohol CoA forming) mcrAAS20429.1 Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1Simmondsia long chain chinensis acyl-CoA 9A 1.2.1.b 4- 4-aminobutanal4-aminobutyryl- sucD P38947.1 Clostridium Succinyl-CoA amino- CoAkluyveri butyryl- reductase (or 4- CoA aminobutanal dehydrogenase) sucDNP_904963.1 Porphyromonas Succinyl-CoA gingivalis Msed_0709 YP_Metallosphaera Malonyl-CoA 001190808.1 sedula 9A 1.1.1.a 4-amino-4-aminobutan- 4-aminobutan- ADH2 NP_014032.1 Saccharymyces generalbutanal 1-ol 1-ol cerevisiae dehydrogenase yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium Succinate kluyveri semialdehyde DSM555 9A 1.4.1.a 4-amino- 4- 4-aminobutan- lysDH AB052732 Geobacilluslysine butan- hydroxybutanal 1-ol stearothermophilus 1-ol oxidoreductase(deaminating) lysDH NP_147035.1 Aeropyrum pernix lysine K1 ldh P0A393Bacillus cereus leucine, isoleucine, valine, 2- aminobutanoate 9A2.6.1.a 4-amino- 4- 4-aminobutan- gabT P22256.1 Escherichia coli4-aminobutyryate butan- hydroxybutanal 1-ol 1-ol transaminase abatP50554.3 Rattus norvegicus 3-amino-2- methylpropionate SkyPYD4ABF58893.1 Saccharomyces beta-alanine kluyveri 9A 1.1.1.a 4-1,4-butanediol 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalhydroxy- dehydrogenase cerevisiae butanal yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium Succinate kluyveri semialdehyde DSM555

FIG. 9B depicts exemplary BDO pathway in which 4-aminobutyrate isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 19, along with exemplary genes encoding these enzymes.

Briefly, 4-aminobutyrate can be converted to[(4-aminobutanolyl)oxy]phosphonic acid by 4-aminobutyrate kinase (EC2.7.2.a). [(4-aminobutanolyl)oxy]phosphonic acid can be converted to4-aminobutanal by 4-aminobutyraldehyde dehydrogenase (phosphorylating)(EC 1.2.1.d). 4-aminobutanal can be converted to 4-aminobutan-1-ol by4-aminobutan-1-ol dehydrogenase (EC 1.1.1.a). 4-aminobutan-1-ol can beconverted to 4-hydroxybutanal by 4-aminobutan-1-ol oxidoreductase(deaminating) (EC 1.4.1.a) or 4-aminobutan-1-ol transaminase (EC2.6.1.a). Alternatively, [(4-aminobutanolyl)oxy]phosphonic acid can beconverted to [(4-oxobutanolyl)oxy]phosphonic acid by[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) (EC1.4.1.a) or [(4-aminobutanolyl)oxy]phosphonic acid transaminase (EC2.6.1.a). [(4-oxobutanolyl)oxy]phosphonic acid can be converted to4-hydroxybutyryl-phosphate by 4-hydroxybutyryl-phosphate dehydrogenase(EC 1.1.1.a). 4-hydroxybutyryl-phosphate can be converted to4-hydroxybutanal by 4-hydroxybutyraldehyde dehydrogenase(phosphorylating) (EC 1.2.1.d). 4-hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).

TABLE 19 BDO pathway from 4-aminobutyrate. Desired Desired GenBank IDKnown Figure EC class substrate product Enzyme name Gene name (ifavailable) Organism Substrate 9B 2.7.2.a 4- [(4- 4-aminobutyrate ackANP_416799.1 Escherichia coli acetate, amino- aminobutanolyl) kinasepropionate butyrate oxy] phosphonic acid buk1 NP_349675 Clostridiumbutyrate acetobutylicum proB NP_414777.1 Escherichia coli glutamate 9B1.2.1.d [(4- 4-aminobutanal 4- asd NP_417891.1 Escherichia coliL-4-aspartyl- amino- aminobutyralde- phosphate butanolyl) hyde oxy]dehydrogenase phosphonic (phosphorylating) acid proA NP_414778.1Escherichia coli L-glutamyl-5- phospate gapA P0A9B2.2 Escherichia coliGlyceraldehyde- 3-phosphate 9B 1.1.1.a 4-amino- 4-aminobutan-4-aminobutan-1- ADH2 NP_014032.1 Saccharymyces general butanal 1-ol ofdehydrogenase cerevisiae yqhD NP_417484.1 Escherichia coli >C3 4hbdL21902.1 Clostridium Succinate kluyveri semialdehyde DSM 555 9B 1.4.1.a4-amino- 4- 4-aminobutan- lysDH AB052732 Geobacillus lysine butan-hydroxybutanal 1-ol stearothermophilus 1-ol oxidoreductase (deaminating)lysDH NP_147035.1 Aeropyrum lysine pernix K1 ldh P0A393 Bacillus cereusleucine, isoleucine, valine, 2- aminobutanoate 9B 2.6.1.a 4-amino- 4-4-aminobutan- gabT P22256.1 Escherichia coli 4-aminobutyryate butan-hydroxybutanal 1-ol 1-ol transaminase abat P50554.3 Rattus norvegicus3-amino-2- methylpropionate SkyPYD4 ABF58893.1 Saccharomycesbeta-alanine kluyveri 9B 1.4.1.a [(4- [(4- [(4- lysDH AB052732Geobacillus lysine amino- oxobutanolyl) aminobutanolyl)stearothermophilus butanolyl) oxy] phosphonic oxy]phosphonic oxy] acidacid phosphonic oxidoreductase acid (deaminating) lysDH NP_147035.1Aeropyrum lysine pernix K1 ldh P0A393 Bacillus cereus leucine,isoleucine, valine, 2- aminobutanoate 9B 2.6.1.a [(4- [(4- [(4- gabTP22256.1 Escherichia coli 4-aminobutyryate amino- oxobutanolyl)aminobutanolyl) butanolyl) oxy] phosphonic oxy] phosphonic oxy] acidacid transaminase phosphonic acid SkyPYD4 ABF58893.1 Saccharomycesbeta-alanine kluyveri serC NP_415427.1 Escherichia coli phosphoserine,phosphohydroxy- threonine 9B 1.1.1.a [(4- 4- 4- ADH2 NP_014032.1Saccharymyces general oxo- hydroxybutyryl- hydroxybutyryl- cerevisiaebutanolyl) phosphate phosphate oxy] dehydrogenase phosphonic acid yqhDNP_417484.1 Escherichia coli >C3 4hbd L21902.1 Clostridium Succinatekluyveri semialdehyde DSM 555 9B 1.2.1.d 4- 4- 4- asd NP_417891.1Escherichia coli L-4-aspartyl- hydroxy- hydroxybutanal hydroxybutyraldephosphate butyryl- hyde phosphate dehydrogenase (phosphorylating) proANP_414778.1 Escherichia coli L-glutamyl-5- phospate gapA P0A9B2.2Escherichia coli Glyceraldehyde- 3-phosphate 9B 1.1.1.a 4-1,4-butanediol 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalhydroxy- dehydrogenase cerevisiae butanal yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium Succinate kluyveri semialdehyde DSM555

FIG. 9C shows an exemplary pathway through acetoacetate.

Example VIII Exemplary BDO Pathways from Alpha-Ketoglutarate

This example describes exemplary BDO pathways from alpha-ketoglutarate.

FIG. 10 depicts exemplary BDO pathways in which alpha-ketoglutarate isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 20, along with exemplary genes encoding these enzymes.

Briefly, alpha-ketoglutarate can be converted toalpha-ketoglutaryl-phosphate by alpha-ketoglutarate 5-kinase (EC2.7.2.a). Alpha-ketoglutaryl-phosphate can be converted to2,5-dioxopentanoic acid by 2,5-dioxopentanoic semialdehyde dehydrogenase(phosphorylating) (EC 1.2.1.d). 2,5-dioxopentanoic acid can be convertedto 5-hydroxy-2-oxopentanoic acid by 2,5-dioxopentanoic acid reductase(EC 1.1.1.a). Alternatively, alpha-ketoglutarate can be converted toalpha-ketoglutaryl-CoA by alpha-ketoglutarate CoA transferase (EC2.8.3.a), alpha-ketoglutaryl-CoA hydrolase (EC 3.1.2.a) oralpha-ketoglutaryl-CoA ligase (or alpha-ketoglutaryl-CoA synthetase) (EC6.2.1.a). Alpha-ketoglutaryl-CoA can be converted to 2,5-dioxopentanoicacid by alpha-ketoglutaryl-CoA reductase (or 2,5-dioxopentanoic aciddehydrogenase) (EC 1.2.1.b). 2,5-Dioxopentanoic acid can be converted to5-hydroxy-2-oxopentanoic acid by 5-hydroxy-2-oxopentanoic aciddehydrogenase. Alternatively, alpha-ketoglutaryl-CoA can be converted to5-hydroxy-2-oxopentanoic acid by alpha-ketoglutaryl-CoA reductase(alcohol forming) (EC 1.1.1.c). 5-hydroxy-2-oxopentanoic acid can beconverted to 4-hydroxybutanal by 5-hydroxy-2-oxopentanoic aciddecarboxylase (EC 4.1.1.a). 4-hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).5-hydroxy-2-oxopentanoic acid can be converted to 4-hydroxybutyryl-CoAby 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation) (EC1.2.1.c).

TABLE 20 BDO pathway from alpha-ketoglutarate. Desired Desired GenBankID Figure EC class substrate product Enzyme name Gene name (ifavailable) Organism Known Substrate 10 2.7.2.a alpha- alpha- alpha- ackANP_416799.1 Escherichia coli acetate, keto- ketoglutaryl- ketoglutarate5- propionate glutarate phosphate kinase buk1 NP_349675 Clostridiumbutyrate acetobutylicum proB NP_414777.1 Escherichia coli glutamate 101.2.1.d alpha- 2,5- 2,5- proA NP_414778.1 Escherichia coli L-glutamyl-keto- dioxopentanoic dioxopentanoic 5-phospate glutaryl- acidsemialdehyde phosphate dehydrogenase (phosphorylating) asd NP_417891.1Escherichia coli L-4-aspartyl- phosphate gapA P0A9B2.2 Escherichia coliGlyceraldehyde- 3-phos- phate 10 1.1.1.a 2,5- 5-hydroxy-2- 2,5- ADH2NP_014032.1 Saccharymyces general dioxo- oxopentanoic dioxopentanoiccerevisiae pentanoic acid acid reductase acid yqhD NP_417484.1Escherichia coli >C3 4hbd L21902.1 Clostridium Succinate kluyveri DSM555 semialdehyde 10 2.8.3.a alpha- alpha- alpha- cat1, cat2, P38946.1,Clostridium succinate, 4- keto- ketoglutaryl- ketoglutarate cat3P38942.2, kluyveri hydroxybutyrate, glutarate CoA CoA EDK35586.1butyrate transferase gctA, gctB CAA57199.1, Acidaminococcus glutarateCAA57200.1 fermentans atoA, atoD P76459.1, Escherichia coli butanoateP76458.1 10 3.1.2.a alpha- alpha- alpha- tesB NP_414986 Escherichia coliadipyl-CoA keto- ketoglutaryl- ketoglutaryl- glutarate CoA CoA hydrolaseacot12 NP_570103.1 Rattus norvegicus butyryl-CoA hibch Q6NVY1.2 Homosapiens 3-hydroxypro- panoyl-CoA 10 6.2.1.a alpha- alpha- alpha- sucCDNP_415256.1, Escherichia coli succinate keto- ketoglutaryl-ketoglutaryl- AAC73 823.1 glutarate CoA CoA ligase (or alpha-ketoglutaryl- CoA synthetase) phl CAJ15517.1 Penicillium phenylacetatechrysogenum bioW NP_390902.2 Bacillus subtilis 6-carboxy- hexanoate 101.2.1.b alpha- 2,5- alpha- sucD P38947.1 Clostridium Succinyl-CoA keto-dioxopentanoic ketoglutaryl- kluyveri glutaryl- acid CoA reductase CoA(or 2,5- dioxopentanoic acid dehydrogenase) Msed_0709 YP_ MetallosphaeraMalonyl-CoA 001190808.1 sedula bphG BAA03892.1 Pseudomonas spAcetaldehyde, Propionaldehyde, Butyraldehyde, Isobutyraldehyde andFormaldehyde 10 1.1.1.a 2,5- 5-hydroxy-2- 5-hydroxy-2- ADH2 NP_014032.1Saccharymyces general dioxo- oxopentanoic oxopentanoic yqhD NP_417484.1cerevisiae >C3 pentanoic acid acid 4hbd L21902.1 Escherichia coliSuccinate acid dehydrogenase Clostridium semialdehyde kluyveri DSM 55510 1.1.1.c alpha- 5-hydroxy-2- alpha- adhE2 AAK09379.1 Clostridiumbutanoyl-CoA keto- oxopentanoic ketoglutaryl- acetobutylicum glutaryl-acid CoA reductase CoA (alcohol forming) mcr AAS20429.1 Chloroflexusmalonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia long chain chinensisacyl-CoA 10 4.1.1.a 5-hydroxy- 4- 5-hydroxy-2- pdc P06672.1 Zymomonas2-oxopentanoic -2- hydroxybutanal oxopentanoic mobilus acid oxopen- acidtanoic decarboxylase acid mdlC P20906.2 Pseudomonas 2-oxopentanoicputida acid pdcl P06169 Saccharomyces pyruvate cerevisiae 10 1.1.1.a 4-1,4-butanediol 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalhydroxy- dehydrogenase cerevisiae butanal yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium Succinate kluyveri semialdehyde DSM555 10 1.2.1.c 5-hydroxy- 4- 5-hydroxy-2- sucA, sucB, NP_415254.1,Escherichia coli Alpha- 2- hydroxybutyryl- oxopentanoic lpd NP_415255.1,ketoglutarate oxopen- CoA acid NP_414658.1 tanoic dehydrogenase acid(decarboxylation) bfmBB, NP_390283.1, Bacillus subtilis 2-keto acidsbfmBAA, NP_390285.1, derivatives bfmBAB, NP_390284.1, of valine, bfmBAB,P21880.1 leucine and pdhD isoleucine Bckdha, NP_036914.1, Rattusnorvegicus 2-keto acids Bckdhb, NP_062140.1, derivatives Dbt, DldNP_445764.1, of valine, NP_955417.1 leucine and isoleucine

Example IX Exemplary BDO Pathways from Glutamate

This example describes exemplary BDO pathways from glutamate.

FIG. 11 depicts exemplary BDO pathways in which glutamate is convertedto BDO. Enzymes of such an exemplary BDO pathway are listed in Table 21,along with exemplary genes encoding these enzymes.

Briefly, glutamate can be converted to glutamyl-CoA by glutamate CoAtransferase (EC 2.8.3.a), glutamyl-CoA hydrolase (EC 3.1.2.a) orglutamyl-CoA ligase (or glutamyl-CoA synthetase) (EC 6.2.1.a).Alternatively, glutamate can be converted to glutamate-5-phosphate byglutamate 5-kinase (EC 2.7.2.a). Glutamate-5-phosphate can be convertedto glutamate-5-semialdehyde by glutamate-5-semialdehyde dehydrogenase(phosphorylating) (EC 1.2.1.d). Glutamyl-CoA can be converted toglutamate-5-semialdehyde by glutamyl-CoA reductase (orglutamate-5-semialdehyde dehydrogenase) (EC 1.2.1.b).Glutamate-5-semialdehyde can be converted to 2-amino-5-hydroxypentanoicacid by glutamate-5-semialdehyde reductase (EC 1.1.1.a). Alternatively,glutamyl-CoA can be converted to 2-amino-5-hydroxypentanoic acid byglutamyl-CoA reductase (alcohol forming) (EC 1.1.1.c).2-Amino-5-hydroxypentanoic acid can be converted to5-hydroxy-2-oxopentanoic acid by 2-amino-5-hydroxypentanoic acidoxidoreductase (deaminating) (EC 1.4.1.a) or 2-amino-5-hydroxypentanoicacid transaminase (EC 2.6.1.a). 5-Hydroxy-2-oxopentanoic acid can beconverted to 4-hydroxybutanal by 5-hydroxy-2-oxopentanoic aciddecarboxylase (EC 4.1.1.a). 4-Hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).Alternatively, 5-hydroxy-2-oxopentanoic acid can be converted to4-hydroxybutyryl-CoA by 5-hydroxy-2-oxopentanoic acid dehydrogenase(decarboxylation) (EC 1.2.1.c).

TABLE 21 BDO pathway from glutamate. Desired Desired GenBank ID FigureEC class substrate product Enzyme name Gene name (if available) OrganismKnown Substrate 11 2.8.3.a glutamate glutamyl- glutamate CoA cat1, cat2,P38946.1, Clostridium succinate, 4- CoA transferase cat3 P38942.2,kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctB CAA57199.1,Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoD P76459.1,Escherichia coli butanoate P76458.1 11 3.1.2.a glutamate glutamyl-glutamyl-CoA tesB NP_414986 Escherichia coli adipyl-CoA CoA hydrolaseacot12 NP_570103.1 Rattus norvegicus butyryl-CoA hibch Q6NVY1.2 Homosapiens 3-hydroxypro- panoyl-CoA 11 6.2.1.a glutamate glutamyl-glutamyl-CoA sucCD NP_415256.1, Escherichia coli succinate CoA ligase(or AAC73 823.1 glutamyl- CoA synthetase) phl CAJ15517.1 Penicilliumphenylacetate chrysogenum bioW NP_390902.2 Bacillus subtilis6-carboxyhexanoate 11 2.7.2.a glutamate glutamate- glutamate ackANP_416799.1 Escherichia coli acetate, propionate 5- 5-kinase phosphatebuk1 NP_349675 Clostridium butyrate acetobutylicum proB NP_414777.1Escherichia coli glutamate 11 1.2.1.d gluta- glutamate- glutamate-5-proA NP_414778.1 Escherichia coli L-glutamyl- mate-5- 5-semi-semialdehyde 5-phospate phosphate aldehyde dehydrogenase (phospho-rylating) asd NP_417891.1 Escherichia coli L-4-aspartyl- phosphate gapAP0A9B2.2 Escherichia coli Glyceraldehyde-3- phosphate 11 1.2.1.bglutamyl- glutamate- glutamyl-CoA sucD P38947.1 Clostridium Succinyl-CoACoA 5-semi- reductase (or kluyveri aldehyde glutamate-5- semialdehydedehydrogenase) Msed_0709 YP_ Metallosphaera Malonyl-CoA 001190808.1sedula bphG BAA03892.1 Pseudomonas sp Acetaldehyde, Propionaldehyde,Butyraldehyde, Isobutyraldehyde and Formaldehyde 11 1.1.1.a gluta-2-amino-5- glutamate-5- ADH2 NP_014032.1 Saccharymyces general mate-5-hydroxy- semialdehyde cerevisiae semi- pentanoic reductase aldehyde acidyqhD NP_417484.1 Escherichia coli >C3 4hbd L21902.1 ClostridiumSuccinate kluyveri DSM 555 semialdehyde 11 1.1.1.c glutamyl- 2-amino-5-glutamyl-CoA adhE2 AAK09379.1 Clostridium butanoyl-CoA CoA hydroxy-reductase acetobutylicum pentanoic (alcohol acid forming) mcr AAS20429.1Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia longchain acyl- chinensis CoA 11 1.4.1.a 2-amino-5- 5-hydroxy- 2-amino-5-gdhA P00370 Escherichia coli glutamate hydroxy- 2-oxopen- hydroxy-penta- tanoic pentanoic noic acid acid acid oxidoreductase (deaminating)ldh P0A393 Bacillus cereus leucine, isoleucine, valine, 2-aminobutanoate nadX NP_229443.1 Thermotoga aspartate maritima 11 2.6.1.a2-amino-5- 5-hydroxy- 2-amino-5- aspC NP_415448.1 Escherichia coliaspartate hydroxy- 2-oxo- hydroxy- penta- pentanoic pentanoic noic acidacid acid transaminase AAT2 P23542.3 Saccharomyces aspartate cerevisiaeavtA YP_026231.1 Escherichia coli valine, alpha- aminobutyrate 114.1.1.a 5-hydroxy- 4- 5-hydroxy-2- pdc P06672.1 Zymomonas 2-oxopentanoic2- hydroxy- oxopentanoic mobilus acid oxopen- butanal acid tanoicdecarboxylase acid mdlC P20906.2 Pseudomonas 2-oxopentanoic putida acidpdc1 P06169 Saccharomyces pyruvate cerevisiae 11 1.1.1.a 4- 1,4-buta-1,4-butanediol ADH2 NP_014032.1 Saccharymyces general hydroxy- nedioldehydrogenase cerevisiae butanal yqhD NP_417484.1 Escherichia coli >C34hbd L21902.1 Clostridium Succinate kluyveri DSM 555 semialdehyde 111.2.1.c 5-hydroxy- 4- 5-hydroxy-2- sucA, sucB, NP_415254.1, Escherichiacoli Alpha- 2- hydroxy- oxopentanoic lpd NP_415255.1, ketoglutarateoxopen- butyryl- acid NP_414658.1 tanoic CoA dehydrogenase acid (decar-boxylation) bfmBB, NP_390283.1, Bacillus subtilis 2-keto acids bfmBAA,NP_390285.1, derivatives bfmBAB, NP_390284.1, of valine, bfmBAB,P21880.1 leucine and pdhD isoleucine Bckdha, NP_036914.1, Rattusnorvegicus 2-keto acids Bckdhb, NP_062140.1, derivatives Dbt,NP_445764.1, of valine, Dld NP_955417.1 leucine and isoleucine

Example X Exemplary BDO from Acetoacetyl-CoA

This example describes an exemplary BDO pathway from acetoacetyl-CoA.

FIG. 12 depicts exemplary BDO pathways in which acetoacetyl-CoA isconverted to BDO. Enzymes of such an exemplary BDO pathway are listed inTable 22, along with exemplary genes encoding these enzymes.

Briefly, acetoacetyl-CoA can be converted to 3-hydroxybutyryl-CoA by3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.a). 3-Hydroxybutyryl-CoAcan be converted to crotonoyl-CoA by 3-hydroxybutyryl-CoA dehydratase(EC 4.2.1.a). Crotonoyl-CoA can be converted to vinylacetyl-CoA byvinylacetyl-CoA Δ-isomerase (EC 5.3.3.3). Vinylacetyl-CoA can beconverted to 4-hydroxybutyryl-CoA by 4-hydroxybutyryl-CoA dehydratase(EC 4.2.1.a). 4-Hydroxybutyryl-CoA can be converted to 1,4-butanediol by4-hydroxybutyryl-CoA reductase (alcohol forming) (EC 1.1.1.c).Alternatively, 4-hydroxybutyryl-CoA can be converted to 4-hydroxybutanalby 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanal dehydrogenase)(EC 1.2.1.b). 4-Hydroxybutanal can be converted to 1,4-butanediol by1,4-butanediol dehydrogenase (EC 1.1.1.a).

TABLE 22 BDO pathway from acetoacetyl-CoA. Desired Desired Gene GenBankID Figure EC class substrate product Enzyme name name (if available)Organism Known Substrate 12 1.1.1.a acetoacetyl- 3- 3-hydroxybutyryl-hbd NP_349314.1 Clostridium 3-hydroxybutyryl- CoA hydroxybutyryl- CoAdehydrogenase acetobutylicum CoA CoA hbd AAM14586.1 Clostridium3-hydroxybutyryl- beijerinckii CoA Msed_ YP_001191505 Metallosphaerapresumed 3- 1423 sedula hydroxybutyryl- CoA 12 4.2.1.a 3- crotonoyl-CoA3-hydroxybutyryl- crt NP_349318.1 Clostridium 3-hydroxybutyryl-hydroxybutyryl- CoA dehydratase acetobutylicum CoA CoA maoC NP_415905.1Escherichia coli 3-hydroxybutyryl- CoA paaF NP_415911.1 Escherichia coli3-hydroxyadipyl- CoA 12 5.3.3.3 crotonoyl-CoA vinylacetyl-vinylacetyl-CoA abfD YP_001396399.1 Clostridium 4-hydroxybutyryl- CoAΔ-isomerase kluyveri CoA DSM 555 abfD P55792 Clostridium4-hydroxybutyryl- aminobutyricum CoA abfD YP_001928843 Porphyromonas4-hydroxybutyryl- gingivalis CoA ATCC 33277 12 4.2.1.a vinylacetyl- 4-4-hydroxybutyryl- abfD YP_001396399.1 Clostridium 4-hydroxybutyryl- CoAhydroxybutyryl- CoA dehydratase kluyveri CoA CoA DSM 555 abfD P55792Clostridium 4-hydroxybutyryl- aminobutyricum CoA abfD YP_001928843Porphyromonas 4-hydroxybutyryl- gingivalis CoA ATCC 33277 12 1.1.1.c 4-1,4-butanediol 4-hydroxybutyryl- adhE2 AAK09379.1 Clostridiumbutanoyl-CoA hydroxybutyryl- CoA reductase acetobutylicum CoA (alcoholforming) mcr AAS20429.1 Chloroflexus malonyl-CoA aurantiacus FARAAD38039.1 Simmondsia long chain chinensis acyl-CoA 12 1.2.1.b 4- 4-4-hydroxybutyryl- sucD P38947.1 Clostridium Succinyl-CoA hydroxybutyryl-hydroxybutanal CoA reductase (or kluyveri CoA 4-hydroxybutanaldehydrogenase) sucD NP_904963.1 Porphyromonas Succinyl-CoA gingivalisMsed_ YP_001190808.1 Metallosphaera Malonyl-CoA 0709 sedula 12 1.1.1.a4- 1,4-butanediol 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalhydroxybutanal dehydrogenase cerevisiae yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium Succinate kluyveri semialdehyde DSM555

Example XI Exemplary BDO Pathway from Homoserine

This example describes an exemplary BDO pathway from homoserine.

FIG. 13 depicts exemplary BDO pathways in which homoserine is convertedto BDO. Enzymes of such an exemplary BDO pathway are listed in Table 23,along with exemplary genes encoding these enzymes.

Briefly, homoserine can be converted to 4-hydroxybut-2-enoate byhomoserine deaminase (EC 4.3.1.a). Alternatively, homoserine can beconverted to homoserine-CoA by homoserine CoA transferase (EC 2.8.3.a),homoserine-CoA hydrolase (EC 3.1.2.a) or homoserine-CoA ligase (orhomoserine-CoA synthetase) (EC 6.2.1.a). Homoserine-CoA can be convertedto 4-hydroxybut-2-enoyl-CoA by homoserine-CoA deaminase (EC 4.3.1.a).4-Hydroxybut-2-enoate can be converted to 4-hydroxybut-2-enoyl-CoA by4-hydroxybut-2-enoyl-CoA transferase (EC 2.8.3.a),4-hydroxybut-2-enoyl-CoA hydrolase (EC 3.1.2.a), or4-hydroxybut-2-enoyl-CoA ligase (or 4-hydroxybut-2-enoyl-CoA synthetase)(EC 6.2.1.a). Alternatively, 4-hydroxybut-2-enoate can be converted to4-hydroxybutyrate by 4-hydroxybut-2-enoate reductase (EC 1.3.1.a).4-Hydroxybutyrate can be converted to 4-hydroxybutyryl-coA by4-hydroxybutyryl-CoA transferase (EC 2.8.3.a), 4-hydroxybutyryl-CoAhydrolase (EC 3.1.2.a), or 4-hydroxybutyryl-CoA ligase (or4-hydroxybutyryl-CoA synthetase) (EC 6.2.1.a). 4-Hydroxybut-2-enoyl-CoAcan be converted to 4-hydroxybutyryl-CoA by 4-hydroxybut-2-enoyl-CoAreductase (EC 1.3.1.a). 4-Hydroxybutyryl-CoA can be converted to1,4-butanediol by 4-hydroxybutyryl-CoA reductase (alcohol forming) (EC1.1.1.c). Alternatively, 4-hydroxybutyryl-CoA can be converted to4-hydroxybutanal by 4-hydroxybutyryl-CoA reductase (or 4-hydroxybutanaldehydrogenase) (EC 1.2.1.b). 4-Hydroxybutanal can be converted to1,4-butanediol by 1,4-butanediol dehydrogenase (EC 1.1.1.a).

TABLE 23 BDO pathway from homoserine. Desired Desired GenBank ID FigureEC class substrate product Enzyme name Gene name (if available) OrganismKnown Substrate 13 4.3.1.a homoserine 4-hydroxy- homoserine aspANP_418562 Escherichia coli aspartate but-2- deaminase enoate aspAP44324.1 Haemophilus aspartate influenzae aspA P07346 Pseudomonasaspartate fluorescens 13 2.8.3.a homoserine homoserine- homoserine CoAcat1, cat2, P38946.1, Clostridium succinate, 4- CoA transferase cat3P38942.2, kluyveri hydroxybutyrate, EDK35586.1 butyrate gctA, gctBCAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoDP76459.1, Escherichia coli butanoate P76458.1 13 3.1.2.a homoserinehomoserine- homoserine-CoA tesB NP_414986 Escherichia coli adipyl-CoACoA hydrolase acot12 NP_570103.1 Rattus norvegicus butyryl-CoA hibchQ6NVY1.2 Homo sapiens 3- hydroxypro- panoyl-CoA 13 6.2.1.a homoserinehomoserine- homoserine-CoA sucCD NP_415256.1, Escherichia coli succinateCoA ligase (or AAC73823.1 homoserine-CoA synthetase) phl CAJ15517.1Penicillium phenylacetate chrysogenum bioW NP_390902.2 Bacillus subtilis6- carboxyhexanoate 13 4.3.1.a homoserine- 4-hydroxy- homoserine-CoAacl1 CAG29274.1 Clostridium beta-alanyl-CoA CoA but-2- deaminasepropionicum enoyl-CoA acl2 CAG29275.1 Clostridium beta-alanyl-CoApropionicum MXAN_ YP_632558.1 Myxococcus beta-alanyl-CoA 4385 xanthus 132.8.3.a 4-hydroxy- 4-hydroxy- 4-hydroxybut-2- cat1, cat2, P38946.1,Clostridium succinate, 4- but- but-2- enoyl-CoA cat3 P38942.2, kluyverihydroxybutyrate, 2-enoate enoyl-CoA transferase EDK35586.1 butyrategctA, gctB CAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentansatoA, atoD P76459.1, Escherichia coli butanoate P76458.1 13 3.1.2.a4-hydroxy- 4-hydroxy- 4-hydroxybut-2- tesB NP_414986 Escherichia coliadipyl-CoA but- but-2- enoyl-CoA 2-enoate enoyl-CoA hydrolase acot12NP_570103.1 Rattus norvegicus butyryl-CoA hibch Q6NVY1.2 Homo sapiens 3-hydroxypro- panoyl-CoA 13 6.2.1.a 4-hydroxy- 4-hydroxy- 4-hydroxybut-2-sucCD NP_415256.1, Escherichia coli succinate but- but-2- enoyl-CoAAAC73823.1 2-enoate enoyl-CoA ligase (or 4-hydroxybut-2- enoyl-CoAsynthetase) phl CAJ15517.1 Penicillium phenylacetate chrysogenum bioWNP_390902.2 Bacillus subtilis 6- carboxyhexanoate 13 1.3.1.a 4-hydroxy-4- 4-hydroxybut-2- enr CAA71086.1 Clostridium but- hydroxy- enoatereductase tyrobutyricum 2-enoate butyrate enr CAA76083.1 Clostridiumkluyveri enr YP_430895.1 Moorella thermoacetica 13 2.8.3.a 4- 4-4-hydroxybutyryl- cat1, cat2, P38946.1, Clostridium succinate, 4-hydroxy- hydroxy- CoA transferase cat3 P38942.2, kluyverihydroxybutyrate, buty- butyryl- EDK35586.1 butyrate rate coA gctA, gctBCAA57199.1, Acidaminococcus glutarate CAA57200.1 fermentans atoA, atoDP76459.1, Escherichia coli butanoate P76458.1 13 3.1.2.a 4- 4-4-hydroxybutyryl- tesB NP_414986 Escherichia coli adipyl-CoA hydroxy-hydroxy- CoA hydrolase buty- butyryl- rate coA acot12 NP_570103.1 Rattusnorvegicus butyryl-CoA hibch Q6NVY1.2 Homo sapiens 3- hydroxypro-panoyl-CoA 13 6.2.1.a 4- 4- 4-hydroxybutyryl- sucCD NP_415256.1,Escherichia coli succinate hydroxy- hydroxy- CoA ligase (or 4-AAC73823.1 buty- butyryl- hydroxybutyryl- rate coA CoA synthetase) phlCAJ15517.1 Penicillium phenylacetate chrysogenum bioW NP_390902.2Bacillus subtilis 6- carboxyhexanoate 13 1.3.1.a 4-hydroxy- 4-4-hydroxybut-2- bcd, etfA, NP_349317.1, Clostridium but- hydroxy-enoyl-CoA etfB NP_349315.1, acetobutylicum 2-enoyl- butyryl- reductaseNP_349316.1 CoA CoA TER Q5EU90.1 Euglena gracilis TDE0597 NP_971211.1Treponema denticola 8 1.1.1.c 4- 1,4-butanediol 4-hydroxybutyryl- adhE2AAK09379.1 Clostridium butanoyl-CoA hydroxy- CoA reductaseacetobutylicum butyryl- (alcohol forming) CoA mcr AAS20429.1Chloroflexus malonyl-CoA aurantiacus FAR AAD38039.1 Simmondsia longchain acyl- chinensis CoA 8 1.2.1.b 4- 4- 4-hydroxybutyryl- sucDP38947.1 Clostridium Succinyl-CoA hydroxy- hydroxy- CoA reductasekluyveri butyryl- butanal (or 4- CoA hydroxybutanal dehydrogenase) sucDNP_904963.1 Porphyromonas Succinyl-CoA gingivalis Msed_0709 YP_Metallosphaera Malonyl-CoA 001190808.1 sedula 8 1.1.1.a 4-1,4-butanediol 1,4-butanediol ADH2 NP_014032.1 Saccharymyces generalhydroxy- dehydrogenase cerevisiae butanal yqhD NP_417484.1 Escherichiacoli >C3 4hbd L21902.1 Clostridium Succinate kluyveri DSM 555semialdehyde

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

1-39. (canceled)
 40. A recombinant microorganism adapted tobiosynthesize 1,4-butanediol (“1,4-BDO”), the recombinant microorganismcomprising one or more nucleic acid sequences encoding one or more of 1)a first polypeptide providing acetyl-CoA acetyltransferase activity, 2)a second polypeptide providing β-hydroxybutyryl-CoA dehydrogenaseactivity; 3) a third polypeptide providing crotonase activity; a 4)fourth polypeptide providing vinylacetyl-CoA-Δ-isomerase and4-hydroxybutyryl-CoA dehydratase activities; 5) a fifth polypeptideproviding 4-hydroxybutyrate-CoA-hydrolase activity, and 6) a sixthpolypeptide providing 1,3-propanediol dehydrogenase activity, whereinthe recombinant microorganism biosynthesizes 1,4-BDO utilizing saidpolypeptides.
 41. The recombinant microorganism of claim 40, wherein theone or more nucleic acid sequences comprise at least one of thiL, hbd,crt, abfD, abfT, and dhaT.
 42. The recombinant microorganism of claim40, wherein the recombinant microorganism is adapted to biosynthesize1,4-BDO by condensing two acetyl-CoA moieties into acetoacetyl-CoA. 43.The recombinant microorganism of claim 42, comprising aldehydedehydrogenase.
 44. (canceled)
 45. A recombinant microorganism adapted tobiosynthesize 1,4-butanediol (“1,4-BDO”), the recombinant microorganismcomprising one or more nucleic acid sequences encoding one or more of 1)a first polypeptide providing a-ketoglutarate decarboxylase activity, 2)a second polypeptide providing 4-hydroxybutyrate dehydrogenase activity,and 3) a third polypeptide providing 1,3-propanediol dehydrogenaseactivity, wherein the recombinant microorganism biosynthesizes 1,4-BDOutilizing said polypeptides.
 46. The recombinant microorganism of claim45, wherein the one or more nucleic acid sequences comprise at least oneof kgd, 4hbd, and dhaT.
 47. The recombinant microorganism of claim 45wherein the recombinant microorganism is adapted to biosynthesize1,4-BDO from citrate, wherein the citrate is derived from oxaloacetateand acetyl-CoA.
 48. The recombinant microorganism of claim 47,comprising: aconitase, isocitrate dehydrogenase, aldehyde dehydrogenase,and methylcitrate synthase; or aconitase, isocitrate dehydrogenase,aldehyde dehydrogenase, and citrate synthase.
 49. (canceled)
 50. Arecombinant microorganism adapted to biosynthesize 1,4-butanediol(“1,4-BDO”), the recombinant microorganism comprising one or morenucleic acid sequences encoding one or more of 1) a first polypeptideproviding fumarase activity, 2) a second polypeptide providing fumaratereductase activity, 3) a third polypeptide providing succinatesemialdehyde dehydrogenase activity, 4) a fourth polypeptide providingone or both of succinyl-CoA synthetase activity and succinatesemialdehyde dehydrogenase activity, 5) a fifth polypeptide providing4-hydroxybutyrate dehydrogenase activity, 6) a sixth polypeptideproviding aldehyde dehydrogenase activity, and 7) a seventh polypeptideproviding 1,3-propanediol dehydrogenase activity, wherein therecombinant microorganism biosynthesizes 1,4-BDO utilizing saidpolypeptides.
 51. The recombinant microorganism of claim 50, wherein theone or more nucleic acid sequences comprise at least one of fumA, fumB,fumC, frd, yneI, sucC, sucD, 4hbD, adh, and dhaT.
 52. The recombinantmicroorganism of claim 50 wherein the recombinant microorganism isadapted to biosynthesize 1,4-BDO from malate, wherein the malate isderived from oxaloacetate and/or from pyruvate.
 53. The recombinantmicroorganism of claim 52, comprising fumarase, succinate semialdehydedehydrogenase, and aldehyde dehydrogenase, or fumarase, succinyl-CoAsynthetase, and aldehyde dehydrogenase. 54-56. (canceled)
 57. Arecombinant microorganism of claim 40 wherein a nucleic acid sequenceencoding one of the indicated polypeptides selectively hybridizes withone of the nucleic acid sequences encoding kgd, 4hbd, dhaT, thiL, crt,bcd, etfB, etfA, hbd, abfD, abfT and yneI. 58-59. (canceled)
 60. Therecombinant microorganism of claim 40, wherein at least one of the oneor more nucleic acid sequences is heterologous.
 61. A method ofbiosynthesis of 1,4-BDO comprising providing in a bioreactor vessel amicroorganism of claim 40, a carbon source, and a media, and conductinga bio-production event under suitable conditions and for a suitable timeto obtain a measurable quantity of 1,4-BDO.
 62. A method of biosynthesisof any of the identified downstream products of 1,4-BDO comprisingpracticing the method of claim 61 and thereafter practicing a method forconversion of 1,4-BDO to one of said identified downstream products. 63.The method of claim 62 wherein the method for conversion comprisesesterification or ester exchange reaction, or dehydration under acidicconditions to form tetrahydrofuran.
 64. (canceled)
 65. A bio-productionsystem comprising a bioreactor vessel, a microorganism of claim 40, acarbon source, and a media, wherein the system is adapted to conduct abio-production event under suitable conditions and for a suitable timeto produce a measurable quantity of 1,4-BDO.
 66. An industrial-scalemicrobial bioreactor system comprising: (a) a bioreactor vessel; (b) acarbon source; (c) a recombinant microorganism of claim 40; and (d) amedia. 67-68. (canceled)
 69. The industrial-scale microbial bioreactorof claim 66, wherein the carbon source is a sugar.
 70. Theindustrial-scale microbial bioreactor of claim 69, wherein the sugar issucrose, glucose, xylose, cellulose or hemixellulose.